Compositions comprising omega-3 fatty acids, 17-hdha and 18-hepe and methods of using same

ABSTRACT

The present invention relates to a polyunsaturated fatty acid composition comprising Omega-3 fatty acids, 17-HDHA and 18-HEPE. The composition can furthermore comprise DPA and/or an acceptable carrier and can be present in a capsule or other suitable dosage unit. The invention also relates to the process of obtaining the composition and methods for using same.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.17/070,401, filed Oct. 14, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/843,416, filed Apr. 8, 2020, which is acontinuation of U.S. patent application Ser. No. 15/757,023, filed Mar.2, 2018 (now U.S. Pat. No. 10,653,703), which is a 35 USC § 371 U.S.National Stage Application of International Patent Application No.PCT/US2016/050397, filed Sep. 6, 2016, which claims priority to U.S.Provisional Application No. 62/213,958, filed Sep. 3, 2015, the entiretyof each which is hereby incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to a polyunsaturated fatty acidcomposition comprising 17-HDHA and 18-HEPE among other Omega-3 fattyacids. The compositions can furthermore comprise DPA and/or anacceptable carrier and can be present in a capsule or other suitabledosage unit. Processes of obtaining and using the compositions are alsoprovided.

BACKGROUND

Inflammation is an unspecific response in defense of external pathogenagents to eliminate them and repair damaged tissues. Inflammation is acomplex physiologic process that could be considered as acute or chronicdepending on the duration of this process.

Chronic inflammation is maintained over time, as a result of a lack ofresolution of the acute initial phase of the inflammatory response orprogressive initiation associated with diseases such as rheumatoidarthritis, atherosclerosis, tuberculosis, cancer, vascular diseases,metabolic syndrome, and neurological diseases as Alzheimer among others.

Resolution of the inflammation is a different process from theanti-inflammatory process. Resolution of inflammation can be defined asthe interval between maximum neutrophil infiltration to the point whenthey are lost from the tissue. Complete resolution is the ideal outcomeof inflammation, although, if not properly regulated, it can lead tochronic inflammation, fibrosis, and loss of function.

Pathologists divide the inflammatory response into initiation andresolution. The natural mechanism of the resolution of inflammation hasacquired a high relevance during the last years due to the inflammationbeing recognized as an important characteristic of the above diseases.Resolution was considered to be a passive process before the discoveryand identification of specialized pro-resolving mediators.

Effective clearance of microbial infections and damaged tissue isself-limited and followed by resolution of inflammation. Resolution canbe defined at the cellular level as the disappearance of accumulatedpolymorphonuclear leukocytes, and at the macroscopic level asreconstitution of tissue architecture and restoration of normalfunction. Complete restoration of tissue integrity after bacterialinfection is directly related to the efficiency of microbe clearance andthen to leukocyte clearance. Several mechanisms appear to drive thedisappearance of inflammatory leukocytes. Apoptosis of leukocytes is oneimportant route of elimination. Once phagocytosis is complete,leukocytes undergo programmed cell deaths in response to locallyreleased mediators which regulate the rate of apoptosis. Aspolymorphonuclear leukocytes die, they simultaneously function ascytokine sinks and sequester earlier released pro-inflammatorycytokines. Apoptotic neutrophils are subsequently phagocytozed bymacrophages (efferocytosis) in a so-called non-phlogistic fashion (i.e.,in the absence of further generation of pro-inflammatory mediators), butwith increased formation of anti-inflammatory mediators such astransforming growth factor-β (TGF-β), lipoxin A4 (LXA4) andinterleukin-10. Another important route of elimination of leukocytes isegress from the inflamed tissue, as shown for eosinophils in pulmonaryinflammation. Macrophages which have eliminated apoptotic neutrophilsdisappear in turn by either apoptosis or egress via the lymphatic systemas inflammation resolves.

Development of new products to facilitate the resolution of theinflammation, especially in chronic diseases associated with animportant inflammatory component, such as Crohn's disease, irritablebowel disease (IBD), fatty liver, wound healing, arterial inflammation,sickle-cell disease, arthritis, psoriasis, urticaria, vasculitis,asthma, ocular inflammation, pulmonary inflammation, dermatitis,cardiovascular diseases, AIDS, Alzheimer's disease, atherosclerosis,cancer, type 2 diabetes, hypertension, infectious diseases,leukemia/lymphoma, metabolic syndrome, neonatology, neuromusculardisorders, obesity, perinatal disorders, rheumatic diseases, stroke,surgical transplantation, vascular disorders, periodontal diseases,brain injury, trauma and neuronal inflammation, among others, is greatlyneeded.

SUMMARY

In several embodiments, the present disclosure provides apolyunsaturated fatty acid composition comprising about 20% to about95%, by weight, Omega-3 fatty acids, and 17-HDHA and 18-HEPE in a totalamount of about 0.0005% to about 1% by weight of the composition. Insome embodiments, the composition can further comprise DPA and/or anacceptable carrier and can be present in a capsule or other suitabledosage unit.

In one embodiment, the present disclosure provides a polyunsaturatedfatty acid composition comprising about 20% to about 95%, by weight,Omega-3 fatty acids, and a collective amount of about 0.0005% to about1% of 17-HDHA and 18-HEPE, by weight.

In another embodiment, the present disclosure provides a dietarysupplement, pharmaceutical product, nutraceutical product, medical food,infant formulae and/or prenatal formulae composition comprising acomposition as disclosed herein.

In another embodiment, the present disclosure provides a process forobtaining a composition as disclosed herein from an oil using a methodselected from chromatography, extraction, distillation and/or vacuumrectification.

In another embodiment, the present disclosure provides a process forobtaining a composition as disclosed herein from a crude oil, theprocess comprising: (i) chemically esterifying crude oil with ethanoland a basic catalyst, at a temperature of about 55° C. to about 75° C.to produce esterified oil, (ii) distilling the esterified oil undervacuum of about 0.01 mbar to 0.6 mbar and at a temperature of about 130°C. to 190° C. for about 10 seconds to about 5 minutes to separate fattyacids shorter than 20 carbon atoms to obtain a distilled esterified oilcomprising about 20% to about 95% by weight EPA and/or DHA, and about0.0005% to about 1% by weight 17-HDHA and 18-HEPE (collectively); and(iii) subjecting the polyunsaturated fatty acid composition obtained instep (ii) to supercritical fluid extraction with CO₂ as a supercriticalfluid at a temperature of about 39° C. to about 46° C. and pressure ofabout 80 bar to about 115 bar to remove oxidation, decomposition and/ordegradation products as oligomers, dimers, polymers and conjugateddienes from the composition.

In another embodiment, the present disclosure provides a process forobtaining a composition as disclosed herein from a concentratedesterified oil, the process comprising: (i) distilling the concentratedesterified oil under vacuum of about 0.01 mbar to 0.6 mbar and at atemperature of about 130° C. to 190° C. for about 10 seconds to about 5minutes to separate fatty acids having fewer than 20 carbon atoms in thecorresponding fatty acid chain to obtain a distilled esterified oilcomprising about 20% to about 95% by weight EPA and/or DHA, and about0.0005% to about 1% by weight 17-HDHA and 18-HEPE (collectively); (ii)subjecting the distilled esterified oil obtained in step (i) tosupercritical fluid extraction with CO₂ as a supercritical fluid at atemperature of about 20° C. to 40° C. and at a pressure of about 80 barto about 115 bar to remove oxidation, decomposition and degradationproducts as oligomers, dimers, polymers and conjugated dienes from thecomposition.

In another embodiment, the present disclosure provides a method ofincreasing phagocytic activity of macrophages in a subject comprisingadministering to the subject a phagocytic activity enhancing amount of acomposition as disclosed herein.

In another embodiment, the present disclosure provides a method ofenhancing macrophage polarization toward a pro-resolution phenotype in asubject comprising administering to the subject a macrophagepolarization-enhancing amount of a composition as disclosed herein.

In another embodiment, the present disclosure provides a method ofresolving inflammation associated with a disease in a subject in needthereof comprising administering to the subject aninflammation-resolving amount of a composition as disclosed herein.

In another embodiment, the present disclosure provides a method ofelevating SPM levels in plasma of a human subject comprisingadministering to the human subject an SPM-elevating amount of acomposition as disclosed herein.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein as a vaccine coadjuvant.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein as a chemotherapeutic coadjuvant.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein in the manufacture of a medicament forincreasing phagocytic activity of macrophages in a subject.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein in the manufacture of a medicament forenhancing macrophage polarization toward a pro-resolution phenotype in asubject.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein in the manufacture of a medicament forresolving inflammation associated with a disease in a subject in needthereof.

In another embodiment, the present disclosure provides use of acomposition as disclosed herein in the manufacture of a medicament forelevating SPM levels in plasma of a human subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Biosynthesis of lipoxins and aspirin-triggered lipoxins fromarachidonic acid.

FIG. 1B: Biosynthesis of E-series resolvins: RvE1 and RvE2 from EPA.

FIG. 1C: Biosynthesis of D series resolvins and aspirin-triggered Dseries resolvins.

FIG. 1D: Biosynthesis of protectins: PD1 (the mono-hydroxylated product17S-hydroxy-DHA), the mono-hydroxylated product 17S-hydroxy-DHA, and thedouble oxygenation product 10S,17S-dihydroxy-DHA, an isomer of NPD1/PD1.

FIG. 1E: Formation of maresins: Mar-1 (7,14S-dihydroxy-DHA); also, anisomer 7S,14S-dihydroxy-DHA, a novel double dioxygenation product fromthis biosynthetic pathway.

FIG. 2: Percentage of increase of phagocytosis over vehicle comparative.This figure shows the comparison of macrophage activation in an in vitromodel of three oil fractions in ethyl ester form compared to the valuesobtained with Mar-1 (1 nM). In this model 100% activity is assigned toMar-1 (1 nM) over phagocytic activity.

FIG. 3: Levels of RvD1 (pg/ml) measured in human plasma samples. Thisfigure shows the values of RvD1 measured in plasma in subjectsadministered a composition containing EPA, DHA, 17-HDHA and 18-HEPEcompared with a placebo.

FIGS. 4A and 4B: Total and average flux values. These figures show ananalysis of bioluminescence total flux of the bioluminescence value(bottomless) and the evolution of medium signal value (bottomless) in aninflammatory murine model administered by gavage krill oil and comparedwith indomethacin and control.

FIGS. 5A and 5B: Total and average flux values. These figures show ananalysis of bioluminescence total flux of the bioluminescence value(bottomless) and the evolution of medium signal value (bottomless) in aninflammatory murine model administered by gavage LM03-3 oil and comparedwith indomethacin and control.

FIGS. 6A and 6B: Total and average flux values. These figures show ananalysis of bioluminescence total flux of the bioluminescence radiationvalue (bottomless) and the evolution of medium radiance value(bottomless) in an inflammatory murine model administered by gavagealgae oil and compared with indomethacin and control.

DETAILED DESCRIPTION

The use of numerical values specified in this application, unlessexpressly indicated otherwise, are stated as approximations through theminimum and maximum values specified within the stated ranges, andpreceded by the word “about.” The disclosure of ranges is intended as acontinuous range including every value between the minimum and maximumvalues recited as well as any ranges that can be formed through suchvalues. The numerical values presented in this application representvarious embodiments of the present invention.

In various embodiments, the present invention provides a polyunsaturatedfatty acid composition comprising about 20% to about 95%, by weight, ofOmega-3 fatty acids and about 0.0005% to about 1%17-HDHA and 18-HEPE(collectively) by weight of the composition.

Omega-3 fatty acids are a family of polyunsaturated fatty acidsconsidered as essential fatty acids, because they cannot be synthesizedby mammals. The main components of this family are EPA(5Z,8Z,11Z,14Z,17Z eicosapentaenoic acid), DHA (4Z,7Z,10Z,13Z,16Z,19Zdocosahexaenoic acid), and ALA (9Z,12Z,15Z octadecatrienoic acid).

Other important components of the family of omega-3 fatty acids are n-3DPA (7Z,10Z,13Z,16Z,19Z-docosapentaenoic acid). The compound n-3 DPA isan elongated metabolite/intermediate of EPA and an intermediate productbetween EPA and DHA.

In various embodiments, the Omega-3 fatty acids in the compositioncomprise EPA and/or DHA in an amount between about 20% to about 95% byweight of the composition.

In various embodiments, the Omega-3 fatty acids in the compositioncomprise EPA in an amount between about 20% to about 95% by weight ofthe composition.

In various embodiments, the Omega-3 fatty acids in the compositioncomprise DHA in an amount between about 20% to about 95% by weight ofthe composition.

In various embodiments, the composition comprises EPA in an amountbetween about 10% to about 26% by weight and DHA in an amount of about30% to about 45% by weight of the composition.

SPMs are a new genus with several families of potent endogenousbioactive products derived from precursors essential fatty acids EPA,DHA, ARA and DPA that are biosynthesized by positional andstereospecific incorporation of one, two or three molecules of molecularoxygen into a polyunsaturated fatty acid using EPA, DHA, ALA and DPA assubstrates into a catalyzed reaction involving fatty acid lipoxygenases,cyclooxygenase type-2, when acetylated by aspirin, and severalcytochrome P450 oxidases.

The SPMs include several families of mediators, lipoxins, resolvins,protectins and maresins. Specific potent members of these familiesinclude among others:

-   -   Lipoxin A4: LXA4;        5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid    -   15-epi-lipoxin A4: 15-epi-LXA4;        (5S,6R,7E,9E,11Z,13E,15R)-5,6,15-trihydroxyicosa-7,9,11,13-tetraenoic        acid    -   Lipoxin B4: LXB4;        5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid    -   15-epi-lipoxin B4: 15-epi-LXB4;        5S,14R,15R-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid    -   Resolvin E1: RvE1;        5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid    -   18S-Resolvin E1: 18S-RvE1;        5S,12R,18S-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid    -   20-hydroxy-Resolvin E1: 20-hydroxy-RvE1;        5S,12R,18S,20-tetrahydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic        acid    -   Resolvin E2: RvE2;        5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid    -   18S-Resolvin E2: 18S-RvE2;        5S,18S-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid    -   18S-Resolvin E3: 18S-RvE3;        17R,18S-dihydroxy-5Z,8Z,11Z,13E,15E-eicosapentaenoic acid    -   18R-Resolvin E3: 18R-RvE3;        17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E-eicosapentaenoic acid    -   Maresin 1: MaR1;        7R,14S-dihydroxy-4Z,8E,10E,12E,16Z,19Z-docosahexaenoic acid    -   7S-Maresin 1: 7S-MaR1;        7S,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid    -   13R,14S-Maresin 2: 13R,14S-MaR2;        13R,14S-dihydroxy-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid    -   14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   Protectin DX: PDX;        10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid    -   14S, 21R-diHDHA:        14S,21R-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   14R,21S-diHDHA: 14R,21        S-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   14R,21R-diHDHA:        14R,21R-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   14S, 21S-diHDHA: 14S,21        S-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   16,17-diHDHA:        16,17S-dihydroxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid    -   16,17-Epoxy-DHA:        16,17-Epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid    -   17S-HDHA: 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid    -   7,8-epoxy-17S-HDHA:        17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid    -   Protectin D1: PD1;        10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid    -   10S,17S-HDHA:        10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid    -   16,17S-diHDHA:        16,17S-dihydroxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid    -   16,17-Epoxy-DHA:        16,17-Epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid    -   Resolvin D1: RvD1;        7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid    -   Resolvin D2: RvD2;        7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic        acid    -   Resolvin D3: RvD3;        4S,11,17S-trihydroxy-5E,7E,9E,13Z,15E,19Z-docosahexaenoic acid    -   Resolvin D4: RvD4;        4S,5,17S-trihydroxy-6E,8E,10E,13E,15Z,19Z-docosahexaenoic acid    -   Resolvin D5: RvD5;        7S,17S-dihydroxy-5Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid    -   Resolvin D6: RvD6;        4S,17S-dihydroxy-5E,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid    -   AT-Resolvin D1: AT-RvD1;        7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid    -   AT-Resolvin D2: AT-RvD2;        7S,16R,17R-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid    -   AT-Resolvin D3: AT-RvD3;        4D,11,17R-trihydroxy-5E,7E,9E,13Z,15E,19Z-docosahexaenoic acid    -   AT-Resolvin D4: AT-RvD4;        4S,5,17R-trihydroxy-6E,8E,10E,13E,15Z,19Z-docosahexaenoic acid    -   10S,17S-HDPAn-6:        10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E-docosapentaenoic acid    -   7,17-HDPAn-6: 7,17-dihydroxy-4Z,8E,10Z,13Z,15E-docosapentaenoic        acid    -   7,14-HDPAn-6: 7,14-dihydroxy-4Z,8E,10Z,12Z,16Z-docosapentaenoic        acid    -   10S,17S-HDPAn-6:        10S,17S-dihydroxy-7Z,11E,13Z,15E,19Z-docosapentaenoic acid    -   7,17-HDPAn-6: 7,17-dihydroxy-8E,10Z,13Z,15E,19Z-docosapentaenoic        acid    -   15S-HETE: 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid    -   15R-HETE: 15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid    -   5S-HEPE: 5S-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid    -   5R-HEPE: 15R-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid    -   11S-HEPE: 11S-hydroxy-5Z,8Z,12E,14Z,17Z-eicosapentaenoic acid    -   11R-HEPE: 11R-hydroxy-5Z,8Z,12E,14Z,17Z-eicosapentaenoic acid    -   12S-HEPE: 12S-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid    -   12R-HEPE: 12R-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid    -   15S-HEPE: 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid    -   15R-HEPE: 15R-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid    -   18S-HEPE: 18S-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid    -   18R-HEPE: 18R-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid    -   4S-HDHA: 4S-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid    -   7S-HDHA: 7S-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-docosahexaenoic acid    -   10S-HDHA: 10S-hydroxy-4Z,7Z,11E,13Z,16Z,19Z-docosahexaenoic acid    -   11S-HDHA: 11S-hydroxy-4Z,7Z,9E,13Z,16Z,19Z-docosahexaenoic acid    -   14S-HDHA: 14S-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   14R-HDHA: 14R-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid    -   17S-HDHA: 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid    -   17R-HDHA: 17R-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid    -   20S-HDHA: 20S-hydroxy-4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid    -   17S-HDPAn-6: 17S-hydroxy-4Z,7Z,10Z,13Z,15E-docosapentaenoic acid    -   14S-HDPAn-6: 14S-hydroxy-4Z,7Z,10Z,12E,16Z-docosapentaenoic acid    -   10S-HDPAn-6: 10S-hydroxy-4Z,7Z,11E,13Z,16Z-docosapentaenoic acid    -   17S-HDPAn-3: 17S-hydroxy-7Z,10Z,13Z,15E,19Z-docosapentaenoic        acid    -   14S-HDPAn-3: 17S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic        acid    -   10S-HDPAn-6: 10S-hydroxy-7Z,11E,13Z,16Z,19Z-docosapentaenoic        acid    -   17-HpDPAn-3: 17-hydroperoxy-8Z,10Z,13Z,15E,19Z-docosapentaenoic        acid    -   RvD1 n-3DPA: 7,8,17-trihydroxy-9,11,13,15E,19Z-docosapentaenoic        acid    -   RvD2n-3DPA: 7,16,17-trihydroxy-8,10,12,14E,19Z-docosapentaenoic        acid    -   RvD5n-3DPA: 7,17-trihydroxy-8E,10,13,15E,19Z-docosapentaenoic        acid    -   PD1 n-3DPA: 10,17-dihydroxy-7Z,11,13,15,19Z-docosapentaenoic        acid    -   PD2n-3DPA: 16,17-dihydroxy-7Z,10,13,14,19Z-docosapentaenoic acid    -   14-HpDHA: 14-hydroperoxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic        acid    -   MaR1 n-3DPA: 7,14-dihydroxy-8,10,12,16Z,19Z-docosapentaenoic        acid    -   MaR2n-3DPA: 13,14-dihydroxy-7Z,9,11,16Z,19Z-docosapentaenoic        acid    -   MaR3n-3DPA: 14,21-dihydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic        acid

Compositions of the present disclosure comprise Omega-3 (e.g., EPAand/or DHA), 17-HDHA and 18-HEPE. In some embodiments, 17-HDHA and18-HEPE are present in a total amount of about 0.0005% to about 1% byweight in the composition.

In some embodiments, the 17-HDHA is present in an amount of about 0.0002wt. % to about 1 wt. %, for example about 0.0002 wt. %, 0.0004 wt. %,0.0006 wt. %, 0.0008 wt. %, 0.001 wt. %, 0.002 wt. %, 0.003 wt. %, 0.004wt. %, 0.005 wt. %, 0.006 wt. %, 0.007 wt. %, 0.008 wt. %, 0.009 wt. %,0.01 wt. %, 0.02 wt. %, 0.03 wt. %, 0.04 wt. %, 0.05 wt. %, 0.06 wt. %,0.07 wt. %, 0.08 wt. %, 0.09 wt. %, 0.1 wt. %, 0.12 wt. %, 0.14 wt. %,0.16 wt. %, 0.18 wt. %, 0.2 wt. %, about 0.22 wt. %, about 0.24 wt. %,about 0.26 wt. %, about 0.28 wt. %, about 0.3 wt. %, about 0.32 wt. %,about 0.34 wt. %, about 0.36 wt. %, about 0.38 wt. %, about 0.4 wt. %,about 0.42 wt. %, about 0.44 wt. %, about 0.46 wt. %, about 0.48 wt. %,about 0.5 wt. %, about 0.52 wt. %, about 0.54 wt. %, about 0.56 wt. %,about 0.58 wt. %, about 0.6 wt. %, about 0.62 wt. %, about 0.64 wt. %,about 0.66 wt. %, about 0.68 wt. %, about 0.7 wt. %, about 0.72 wt. %,about 0.74 wt. %, about 0.76 wt. %, about 0.78 wt. %, about 0.8 wt. %,about 0.82 wt. %, about 0.84 wt. %, about 0.86 wt. %, about 0.88 wt. %,about 0.9 wt. %, about 0.92 wt. %, about 0.94 wt. %, about 0.96 wt. %,about 0.98 wt. %, or about 1 wt. %.

In some embodiments, the 18-HEPE is present in an amount of about forexample about 0.0002 wt. %, 0.0004 wt. %, 0.0006 wt. %, 0.0008 wt. %,0.001 wt. %, 0.002 wt. %, 0.003 wt. %, 0.004 wt. %, 0.005 wt. %, 0.006wt. %, 0.007 wt. %, 0.008 wt. %, 0.009 wt. %, 0.01 wt. %, 0.02 wt. %,0.03 wt. %, 0.04 wt. %, 0.05 wt. %, 0.06 wt. %, 0.07 wt. %, 0.08 wt. %,0.09 wt. %, 0.1 wt. %, 0.12 wt. %, 0.14 wt. %, 0.16 wt. %, 0.18 wt. %,0.2 wt. %, about 0.22 wt. %, about 0.24 wt. %, about 0.26 wt. %, about0.28 wt. %, about 0.3 wt. %, about 0.32 wt. %, about 0.34 wt. %, about0.36 wt. %, about 0.38 wt. %, about 0.4 wt. %, about 0.42 wt. %, about0.44 wt. %, about 0.46 wt. %, about 0.48 wt. %, about 0.5 wt. %, about0.52 wt. %, about 0.54 wt. %, about 0.56 wt. %, about 0.58 wt. %, about0.6 wt. %, about 0.62 wt. %, about 0.64 wt. %, about 0.66 wt. %, about0.68 wt. %, about 0.7 wt. %, about 0.72 wt. %, about 0.74 wt. %, about0.76 wt. %, about 0.78 wt. %, about 0.8 wt. %, about 0.82 wt. %, about0.84 wt. %, about 0.86 wt. %, about 0.88 wt. %, about 0.9 wt. %, about0.92 wt. %, about 0.94 wt. %, about 0.96 wt. %, about 0.98 wt. %, orabout 1 wt. %.

In some embodiments, the composition further comprises DPA. In someembodiments, the DPA is present in an amount of about 1 wt. % to about10 wt. %, for example about 1 wt. %, about 2 wt. %, about 3 wt. %, about4 wt. %, about 5 wt. %, about 6 wt. %, about 7 wt. %, about 8 wt. %,about 9 wt. %, or about 10 wt. %.

In some embodiments, the composition further comprises 14-HDHA. In someembodiments, the 14-HDHA is present in an amount of about 0.001 wt. % toabout 0.1 wt. %, for example about 0.001 wt. %, about 0.002 wt. %, about0.003 wt. %, about 0.004 wt. %, about 0.005 wt. %, about 0.006 wt. %,about 0.007 wt. %, about 0.008 wt. %, about 0.009 wt. %, about 0.01 wt.%, about 0.02 wt. %, about 0.03 wt. %, about 0.04 wt. %, about 0.05 wt.%, about 0.06 wt. %, about 0.07 wt. %, about 0.08 wt. %, about 0.09 wt.%, or about 0.1 wt. %.

In some embodiments, the composition further comprises 15-HETE. In someembodiments, the 15-HETE is present in an amount of about 0.0001 wt. %to about 0.01 wt. %, for example about 0.0001 wt. %, about 0.0002 wt. %,about 0.0003 wt. %, about 0.0004 wt. %, about 0.0005 wt. %, about 0.0006wt. %, about 0.0007 wt. %, about 0.0008 wt. %, about 0.0009 wt. %, about0.001 wt. %, about 0.0015 wt. %, about 0.002 wt. %, about 0.0025 wt. %,about 0.003 wt. %, about 0.0035 wt. %, about 0.004 wt. %, about 0.0045wt. %, about 0.005 wt. %, about 0.0055 wt. %, about 0.006 wt. %, about0.0065 wt. %, about 0.007 wt. %, about 0.0075 wt. %, about 0.008 wt. %,about 0.0085 wt. %, about 0.009 wt. %, about 0.0095 wt. %, or about 0.01wt. %.

In some embodiments, the composition further comprises 5-HETE. In someembodiments, the 5-HETE is present in an amount of about 0.0001 wt. % toabout 0.01 wt. %, for example about 0.0001 wt. %, about 0.0002 wt. %,about 0.0003 wt. %, about 0.0004 wt. %, about 0.0005 wt. %, about 0.0006wt. %, about 0.0007 wt. %, about 0.0008 wt. %, about 0.0009 wt. %, about0.001 wt. %, about 0.0015 wt. %, about 0.002 wt. %, about 0.0025 wt. %,about 0.003 wt. %, about 0.0035 wt. %, about 0.004 wt. %, about 0.0045wt. %, about 0.005 wt. %, about 0.0055 wt. %, about 0.006 wt. %, about0.0065 wt. %, about 0.007 wt. %, about 0.0075 wt. %, about 0.008 wt. %,about 0.0085 wt. %, about 0.009 wt. %, about 0.0095 wt. %, or about 0.01wt. %.

In some embodiments, the composition further comprises MaR-1. In someembodiments, the MaR-1 is present in an amount of about 0.0001 wt. % toabout 0.01 wt. %, for example about 0.0001 wt. %, about 0.0002 wt. %,about 0.0003 wt. %, about 0.0004 wt. %, about 0.0005 wt. %, about 0.0006wt. %, about 0.0007 wt. %, about 0.0008 wt. %, about 0.0009 wt. %, about0.001 wt. %, about 0.0015 wt. %, about 0.002 wt. %, about 0.0025 wt. %,about 0.003 wt. %, about 0.0035 wt. %, about 0.004 wt. %, about 0.0045wt. %, about 0.005 wt. %, about 0.0055 wt. %, about 0.006 wt. %, about0.0065 wt. %, about 0.007 wt. %, about 0.0075 wt. %, about 0.008 wt. %,about 0.0085 wt. %, about 0.009 wt. %, about 0.0095 wt. %, or about 0.01wt. %.

In some embodiments, the composition comprises pro-inflammatorycompounds in a combined (e.g., total) amount of not more than about0.005 wt. %, for example no more than about 0.005 wt. %, about 0.004 wt.%, about 0.003 wt. %, about 0.002 wt. %, about 0.001 wt. %, about 0.0005wt. %, or no more than about 0.0001 wt. %.

In some embodiments, the composition comprises 12-HETE, LTB4,Prostaglandin E2, Prostaglandin D2, Thromboxane B2, ProstaglandinF_(2α), 20-COOH TLB4, 20-Hydoxy-LTB4, 11-HETE, 6-keto PGF_(1α), and15-keto PGE2 in a combined (e.g., total) amount of not more than about0.005 wt. %, for example no more than about 0.005 wt. %, about 0.004 wt.%, about 0.003 wt. %, about 0.002 wt. %, about 0.001 wt. %, about 0.0005wt. %, or no more than about 0.0001 wt. %.

In some embodiments, the composition comprises a total amount of SPMsand SPM Precursors of about 0.01 wt. % to about 1 wt. %, for exampleabout 0.01 wt. %, about 0.015 wt. %, about 0.02 wt. %, about 0.025 wt.%, about 0.03 wt. %, about 0.035 wt. %, about 0.04 wt. %, about 0.045wt. %, about 0.05 wt. %, about 0.055 wt. %, about 0.06 wt. %, about0.065 wt. %, about 0.07 wt. %, about 0.075 wt. %, about 0.08 wt. %,about 0.085 wt. %, about 0.09 wt. %, about 0.095 wt. %, about 0.1 wt. %,about 0.15 wt. %, about 0.2 wt. %, about 0.25 wt. %, about 0.3 wt. %,about 0.35 wt. %, about 0.4 wt. %, about 0.45 wt. %, about 0.5 wt. %,about 0.55 wt. %, about 0.6 wt. %, about 0.65 wt. %, about 0.7 wt. %,about 0.75 wt. %, about 0.8 wt. %, about 0.85 wt. %, about 0.9 wt. %,about 0.95 wt. %, or about 1 wt. %

In some embodiments, the composition comprises Omega-3 fatty acids orderivatives thereof (e.g., an ester such as an ethyl ester) in a totalamount of about 20 wt. % to about 99 wt. %, for example about 20 wt. %,about 21 wt. %, about 22 wt. %, about 23 wt. %, about 24 wt. %, about 25wt. %, about 26 wt. %, about 27 wt. %, about 28 wt. %, about 29 wt. %,about 30 wt. %, about 31 wt. %, about 32 wt. %, about 33 wt. %, about 34wt. %, about 35 wt. %, about 36 wt. %, about 37 wt. %, about 38 wt. %,about 39 wt. %, about 40 wt. %, about 41 wt. %, about 42 wt. %, about 43wt. %, about 44 wt. %, about 45 wt. %, about 46 wt. %, about 47 wt. %,about 48 wt. %, about 49 wt. %, about 50 wt. %, about 51 wt. %, about 52wt. %, about 53 wt. %, about 54 wt. %, about 55 wt. %, about 56 wt. %,about 57 wt. %, about 58 wt. %, about 59 wt. %, about 60 wt. %, about 61wt. %, about 62 wt. %, about 63 wt. %, about 64 wt. %, about 65 wt. %,about 66 wt. %, about 67 wt. %, about 68 wt. %, about 69 wt. %, about 70wt. %, about 71 wt. %, about 72 wt. %, about 73 wt. %, about 74 wt. %,about 75 wt. %, about 76 wt. %, about 77 wt. %, about 78 wt. %, about 79wt. %, about 80 wt. %, about 81 wt. %, about 82 wt. %, about 83 wt. %,about 84 wt. %, about 85 wt. %, about 86 wt. %, about 87 wt. %, about 88wt. %, about 89 wt. %, about 90 wt. %, about 91 wt. %, about 92 wt. %,about 93 wt. %, about 94 wt. %, about 95 wt. %, about 96 wt. %, about 97wt. %, about 98 wt. %, about 99 wt. %, or greater than about 99 wt. %.

The Omega-3 fatty acids of the composition in a preferred embodiment areEPA and/or DHA, and in a more preferred embodiment, DHA is present in anamount of about 30% to about 45% and EPA is present in an amount ofabout 10% to about 26% by weight and 17-HDHA is present in an amount ofabout 0.0004% to about 0.04%, and 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition.

Omega-3 fatty acids, 17-HDHA and 18-HEPE can be obtained from differentsources including vegetables, microbial, animal or combinations of thesesources, including different oils as, for example, fish oil, krill oil,vegetable oil, microbial oil or combinations among other. In oneembodiment, the Omega-3, 17-HDHA and 18-HEPE are obtained from fish oil,Krill oil and/or algae oil.

The chemical form of the Omega-3, 17-HDHA and 18-HEPE is selected fromfree fatty acids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and combinations thereof. In one embodiment, the Omega-3compounds are in ethyl ester form.

The composition is useful for a pharmaceutical composition, a dietarysupplement, a nutraceutical product, a medical food composition, anutritional composition for infant formulae and/or prenatal formulaethat increases tissue levels of SPM in vivo.

In one embodiment, the composition is present in a capsule or othersuitable dosage unit.

The composition can be obtained using a method selected fromchromatography, extraction, distillation and/or vacuum rectificationfrom several sources as, for example, fish oil and krill oil.

In one embodiment, the distillation is a short path moleculardistillation process.

In several embodiments, the composition can be obtained from a crude oilusing a process comprising several steps. The first step is a chemicallyesterification of the crude oil with ethanol and a basic catalyst at atemperature of about 55° C. to about 85° C. to produce an esterifiedoil.

This esterified oil is distilled to separate fatty acids shorter than 20carbon atoms. This distillation is under vacuum of about 0.01 mbar toabout 0.6 mbar and temperature of about 130° C. to about 190° C. forabout 10 seconds to about 5 minutes to obtain a polyunsaturated fattyacid composition comprising about 20% to about 95% by weight EPA and/orDHA, and about 0.0005% to about 1% 17-HDHA and 18-HEPE by weight. Thisprocess can be covered in several stages in series.

In one embodiment, if necessary, after elimination of fatty acidsshorter than 20 carbon atoms, the esterified oil can be distilled toeliminate fatty acids higher than 23 carbon atoms.

Before the step of distillation, to improve the quality of thecomposition and to remove oxidation, decomposition and/or degradationproducts, the polyunsaturated fatty acid composition is subjected to asupercritical fluid extraction (SFE) with CO₂ as supercritical fluid,pressure of about 80 bar to about 115 bar, and temperature of about 39°C. to about 46° C.

The oxidation, degradation and/or decomposition products eliminated bySFE can be selected from oligomers, dimers, polymers and conjugateddienes among others.

In various embodiments, the polyunsaturated composition is subjected toa step of bleaching under vacuum with bleaching earths and diatomaceousearths.

In an embodiment, the polyunsaturated oil composition is subjected to atransesterification step to obtain a composition containing a mixture oftri-glycerides and partial glycerides (mono-glycerides anddi-glycerides). In one embodiment, the transesterification reaction canbe catalyzed by lipases.

In various embodiments, polyunsaturated oil composition is subjected toa deodorization step under vacuum and a countercurrent flow of nitrogenor water steam.

In one embodiment, the composition is obtained from a starting materialconsisting of esterified concentrated oil, in these embodiments it isnot necessary to make an esterification step.

The concentrated esterified oil is distilled to separate fatty acidsshorter than 20 carbon atoms. This distillation is under vacuum of about0.01 mbar to about 0.6 mbar and temperature of about 130° C. to about190° C. for about 10 seconds to about 5 minutes to obtain apolyunsaturated fatty acid composition comprising about 20% to about 95%by weight EPA and/or DHA, and about 0.0005% to about 1% 17-HDHA and18-HEPE by weight. This process can be covered in several stages inseries.

In one embodiment, if necessary, after elimination of fatty acidsshorter than 20 carbon atoms, the esterified oil can be distilled toeliminate fatty acids higher than 23 carbon atoms.

Before the step of distillation, to improve the quality of thecomposition and to remove oxidation, decomposition and/or degradationproducts, the polyunsaturated fatty acid composition is subjected to asupercritical fluid extraction (SFE) with CO₂ as supercritical fluid,pressure of about 80 bar to about 115 bar, and temperature of about 39°C. to about 46° C.

In one embodiment, the oxidation, degradation and/or decompositionproducts eliminated by SFE can be selected from oligomers, dimers,polymers and conjugated dienes, among others.

In one embodiment, the polyunsaturated oil composition, if necessary,can be subjected to a step of bleaching under vacuum with bleachingearths and diatomaceous earths.

In one embodiment, the polyunsaturated oil composition, if necessary,can be subjected to a transesterification to obtain a compositioncontaining a mixture of tri-glycerides and partial glycerides(mono-glycerides and di-glycerides). In one embodiment, thetransesterification can be made by lipases, such as lipase from Candidaantarctica.

In one embodiment, if necessary, the polyunsaturated oil composition canbe subjected to a deodorization step under vacuum and a countercurrentflow of nitrogen or water steam.

In various embodiments, the composition is useful in a method toincrease phagocytic activity, comprising administering to a subject aphagocytic activity enhancing amount of the composition. In a preferredembodiment, the composition is useful in a method to enhance macrophagepolarization toward an M2-like phenotype in a subject, comprisingadministering to the subject a macrophage polarization enhancing amountof the composition.

In one embodiment, the composition is useful for resolving inflammationassociated with a disease in a subject, administering to the subject aninflammation-resolving amount of the composition.

The diseases can be selected from Crohn's disease, irritable boweldisease (IBD), fatty liver, wound healing, arterial inflammation,sickle-cell disease, arthritis, psoriasis, urticaria, vasculitis,asthma, ocular inflammation, pulmonary inflammation, dermatitis,cardiovascular diseases, AIDS, Alzheimer's disease, atherosclerosis,cancer, type 2 diabetes, hypertension, infectious diseases,leukemia/lymphoma, metabolic syndrome, neonatology, neuromusculardisorders, obesity, perinatal disorders, rheumatic diseases, stroke,surgical transplantation, vascular disorders, periodontal diseases, andbrain injury.

In other embodiments, the composition can be used as a vaccinecoadjuvant and/or as a chemotherapeutic coadjuvant.

In various embodiments, the composition comprises EPA in an amountbetween about 10% to about 26% by weight and DHA in an amount of about30% to about 45% by weight, and 17-HDHA and 18-HEPE in a collectiveamount of about 0.0005% to about 1% by weight of the composition.

In one embodiment, the composition comprises EPA in an amount betweenabout 10% to about 26% by weight and DHA in an amount of about 30% toabout 45% by weight, 17-HDHA in an amount of about 0.0004% to about0.04% by weight of the composition, and 18-HEPE in an amount of about0.0003% to about 0.04% by weight of the composition.

The composition can further comprise DPA. In one embodiment, thecomposition comprises EPA and/or DHA and DPA in an amount between about20% to about 95%, and 17-HDHA and 18-HEPE in an amount of about 0.0005%to about 1% by weight of the composition.

In some embodiments, the composition comprises EPA, DHA and DPA in anamount between about 20% to about 95%, and 17-HDHA and 18-HEPE in acollective amount of about 0.0005% to about 1% by weight of thecomposition.

In various embodiments, the composition also contains at least oneadditional SPM, such as 18R/S-HEPE, 17R/S-HDHA, 5S-HEPE, 15R/S-HEPE,4R/S-HDHA, 7R/S-HDHA, 10R/S-HDHA, 14R/S-HDHA, and/or RvE1.

The Omega-3 fatty acid content in the composition is determined on aweight/weight percent basis relative to all fatty acids present in thecomposition as determined by methods such as those disclosed in theEuropean Pharmacopeia monograph for Omega-3 fatty acids, EuropeanPharmacopeia monograph method 2.49.29 or any equivalent method using gaschromatography, HPLC, FPLC, or other chromatographic method, and suchcontent is expressed as a percentage in FFA content.

17-HDHA and 18-HEPE content of the composition is determined on aweight/weight basis relative to all fatty acids present in thecomposition using liquid chromatography-tandem mass spectrometryemploying diagnostic transitions and co-elution with synthesizeddeuterated standards of 17-HDHA and 18-HEPE.

In some embodiments, the composition further comprises an acceptablecarrier or excipient that may be administered by a variety of routes,for example, oral, topical, transdermal, parenteral, intravenous,intramuscular, rectal, sublingual, epidural, intracerebral, intraocular,subcutaneous, vaginal, transmucosal, intrathecal or intraarticular,among other acceptable carriers or excipients known to those of skill inthe art.

The composition might also include one or more active ingredients, suchas aspirin, curcumin, polyphenols, lutein, astaxanthin, and severalvitamins (including vitamin C and vitamin E), among others.

In some embodiments, the composition can contain an antioxidant toimprove stability of the composition.

In some embodiments, 17-HDHA and 18-HEPE can act as antioxidants in thecomposition.

The composition can be delivered in a variety of forms, such ascapsules, pills, tablets, a powder, sachets, emulsions, suspensions,solutions, sprays for intranasal administration, aerosols, gels, softand hard gelatin capsules, liposomes, chewable tablets, microspheredelivery systems, creams, sterile injectable solutions, an osmoticdelivery system, dry powder inhalers, orally disintegrating tablets,oral sprays, hydrogels, dermal patches, transdermal patches, lotions, orsyrups, among other delivery forms known in the art.

In some embodiments, the composition can be present in a capsule orother suitable dosage unit to be taken orally by a subject.

In some embodiments, the composition can be present in a soft or hardgelatin capsule with different sizes, shapes, colors and forms toaddress bioavailability enhancement, composition stability and othercomposition challenges.

In some embodiments, the composition can be delivered in an emulsionsuitable for oral and/or parenteral administration to a subject. Such anemulsion can simplify the daily intake to just one sachet or spoonserving in oral administration and can also ensure minimal oxidation andgood bioavailability of EPA, DHA, 17-HDHA and 18-HEPE in thecomposition, as well as increase the shelf life of the composition.Furthermore, parenteral nutrition can improve nutrient delivery tocritically ill subjects.

EPA, DHA and DPA are commonly found in marine oils, including fish,algae and krill oil, among other oils, in contrast, ALA is commonlyfound in seeds, such as flax seeds, camelina seeds, and chia seeds,among others. These are not the exclusive sources of Omega-3, however:it can also be obtained from sources as vegetable, microbial, andanimal, or combinations thereof.

In some embodiments, the Omega-3 fatty acids are obtained from ananimal, vegetable and/or microbial source, alongside combinations ofOmega-3 fatty acids obtained from different sources.

In some embodiments, the Omega-3 fatty acids are obtained from fish oil,krill oil, vegetable oil, microbial oil and/or combinations thereof.

In one embodiment, Omega-3 fatty acids are obtained from fish oil.

In one embodiment, Omega-3 fatty acids are obtained from krill oil.

In one embodiment, Omega-3 fatty acids are obtained from algae.

As described in WO 2013/170006, which is incorporated herein byreference in its entirety, SPMs, including 17-HDHA and 18-HEPE, can befound in different natural sources as fish, krill, algae or otherorganisms containing Omega-3 fatty acids.

In some embodiments, 17-HDHA and 18-HEPE are obtained from animal,vegetable and microbial sources.

In some embodiments, 17-HDHA and 18-HEPE are obtained from fish oil,krill oil, vegetable oil, microbial oil and combinations thereof.

In one embodiment, the 17-HDHA and 18-HEPE are obtained from fish oil.

In one embodiment, the 17-HDHA and 18-HEPE are obtained from krill oil.

In one embodiment, the 17-HDHA and 18-HEPE are obtained from algae.

Omega-3 fatty acids, 17-HDHA and 18-HEPE can be present in thecomposition in different chemical forms, including free fatty acids,esters, phospholipids, mono-glycerides, di-glycerides, tri-glycerides,and combinations thereof.

In an embodiment, Omega-3 fatty acids are in ethyl ester form in thecomposition.

In an embodiment, the 17-HDHA and 18-HEPE are in ethyl ester form in anamount of at least 80% by weight, and in a mixture of partial glycerides(tri-glycerides, mono-glycerides and di-glycerides).

In one embodiment, EPA, DHA, 17-HDHA and 18-HEPE are in ethyl esterform.

The composition can be obtained using a variety of methods, for example,chromatography, extraction, supercritical extraction, distillation andvacuum rectification, and/or any other method generally known to thoseskilled in the art of isolating and purifying Omega-3 polyunsaturatedfatty acids.

The composition can be obtained from two different starting materialsconsisting in a crude oil or an esterified concentrated oil.

In various embodiments, the crude oil or the esterified concentrated oilis obtained from marine oil, vegetable oil, microbial oil, and mixturesof these oils.

In various embodiments, the crude oil or the esterified concentratedmarine oil is fish oil and/or krill oil.

In one embodiment, the crude oil or the esterified concentrated oil isobtained from fish oil.

In one embodiment, the oils can be obtained from anchovy, sardine, Jackmackerel, mackerel, tuna, salmon, Pollock, krill and/or algae.

In one embodiment, the crude oil or the esterified concentrated oil isobtained from krill oil.

In one embodiment, the crude oil or the esterified concentrated oil isobtained from algae oil.

In various embodiments, when a crude oil is used as a starting material,the first step is a chemical esterification of the oil with ethanol anda basic catalyst, at a temperature of about 55° C. to about 85° C., toproduce an esterified oil.

In an embodiment, the basic catalyst used in the chemical esterificationis sodium ethoxide (EtONa).

In an embodiment, the basic catalyst used in the chemical esterificationis potassium hydroxide (KOH).

This esterified oil is distilled to separate fatty acids shorter than 20carbon atoms. This distillation is under vacuum of about 0.01 mbar toabout 0.6 mbar and temperature of about 130° C. to about 190° C. forabout 10 seconds to about 5 minutes to obtain a polyunsaturated fattyacid composition comprising about 20% to about 95% by weight EPA and/orDHA, and about 0.0005% to about 1% 17-HDHA and 18-HEPE by weight.

In one embodiment, if necessary, after elimination of fatty acidsshorter than 20 carbon atoms, the esterified oil can be distilled toeliminate fatty acids higher than 23 carbon atoms.

In one embodiment, the distillation is a short path moleculardistillation process.

These distillation steps to eliminate fatty acids shorter than 20 carbonatoms can be covered in several stages in series, until apolyunsaturated fatty acid composition comprising the desiredcomposition is obtained.

Before the step of distillation, to improve the quality of thecomposition and to remove oxidation, decomposition and/or degradationproducts, the polyunsaturated fatty acid composition is subjected to asupercritical fluid extraction (SFE) with CO₂ as supercritical fluid,pressure of about 80 bar to about 115 bar, and temperature of about 39°C. to about 46° C.

In an embodiment, the oxidation, decomposition and/or degradationproducts eliminated by SFE comprise one or more of oligomers, dimers,polymers and conjugated dienes.

In an embodiment, the process, if necessary, can further comprise a stepof bleaching the polyunsaturated fatty acid composition under vacuumwith bleaching earths and diatomaceous earths.

In an embodiment, the process further comprises a step oftransesterification of the polyunsaturated fatty acid composition toobtain a composition containing a mixture of tri-glycerides and partialglycerides (mono-glycerides and di-glycerides).

This transesterification yields a composition comprising a mixture oftri-glycerides and partial glycerides (mono-glycerides anddi-glycerides).

In an embodiment, this transesterification is catalyzed by lipases.

In a preferred embodiment, lipases are active as of about 25° C., withan optimum temperature of about 50° C. to about 70° C.

In one embodiment, the reaction of transesterification is carried outunder vacuum.

In an embodiment, the polyunsaturated oil composition, if necessary, canbe subjected to a deodorization step under vacuum and a countercurrentflow of nitrogen or water steam.

In a preferred embodiment, the deodorization step under vacuum isperformed at a temperature of about 130° C. to about 200° C.

When the starting material is esterified concentrated oil, the processis the same as that described above, but without the first step ofesterification of the starting material.

In various embodiments, this concentrated oil is distilled to separatefatty acids shorter than 20 carbon atoms. This distillation is undervacuum of about 0.01 mbar to about 0.6 mbar and temperature of about130° C. to about 190° C. for about 10 seconds to about 5 minutes toobtain a polyunsaturated fatty acid composition comprising about 20% toabout 95% by weight EPA and/or DHA, and about 0.0005% to about 1%17-HDHA and 18-HEPE by weight.

These distillation steps to eliminate fatty acids shorter than 20 carbonatoms can be covered in several stages in series, until apolyunsaturated fatty acid composition comprising the desiredcomposition is obtained.

In one embodiment, if necessary, after elimination of fatty acidsshorter than 20 carbon atoms, the concentrated oil can be distilled toeliminate fatty acids higher than 23 carbon atoms.

In one embodiment, the distillation is a short path moleculardistillation process.

Before the step of distillation, to improve the quality of thecomposition and to remove oxidation, decomposition and/or degradationproducts, the polyunsaturated fatty acid composition is subjected to asupercritical fluid extraction (SFE) with CO₂ as supercritical fluid,pressure of about 80 bar to about 115 bar, and temperature of about 39°C. to about 46° C.

In an embodiment, the process, if necessary, can further comprise a stepof bleaching the polyunsaturated fatty acid composition under vacuumwith bleaching earths and diatomaceous earths.

In an embodiment, the process further comprises a step oftransesterification of the polyunsaturated fatty acid composition toobtain a composition containing a mixture of tri-glycerides and partialglycerides (mono-glycerides and di-glycerides).

This transesterification yields a mixture of tri-glycerides and partialglycerides (mono-glycerides and di-glycerides).

In an embodiment, this transesterification is catalyzed by lipases.

In one embodiment, lipases are active as of about 25° C., with anoptimum temperature of about 50° C. to about 70° C.

In one embodiment, the reaction of transesterification is carried outunder vacuum.

In an embodiment, the polyunsaturated oil composition, if necessary, canbe subjected to a deodorization step under vacuum and a countercurrentflow of nitrogen or water steam.

In a preferred embodiment, the deodorization step under vacuum isperformed at a temperature of about 130° C. to about 200° C.

Phagocytosis is an important physiological process characterized by theingestion of foreign particles and killing of microorganisms byphagocytic leukocytes (granulocytes, monocytes and macrophages).Phagocytosis involves a complex series of events including, for example,production of pro- and anti-inflammatory cytokines and chemokines.Deficiencies in phagocytosis cause several pathological conditions(i.e., chronic inflammatory diseases) and cause severe and recurrentmicrobial (bacterial and fungal) infections.

The activation of non-phlogistic (non-fever causing) phagocytosis andpolarization of macrophages toward an M2-like phenotype is an essentialstep for the resolution of the inflammatory response and completehomeostasis of adipose tissue. Several SPMs, including RvD1, RvD2,Protectin D1, and Mar-1 (maresin 1), have been established as mediatorsthat stimulate macrophage switching to the M2 phenotype.

In some embodiments, the composition is useful to increase phagocyticactivity of macrophages in a subject, administering to the subject aphagocytic activity enhancing amount of a composition.

When comparing the activation of macrophages through this applicationwith Omacor and a 3624 EE, the results show that Omacor and 3624 EEreduce the activation of macrophages; instead, the composition ofinterest produces an increase of phagocytic activity and polarization ofmacrophages toward a pro-resolution state (i.e., M2) (shown in example2).

In one embodiment, the increase of the phagocytic activity of thecomposition is at least 55% of the maximum value obtained for Mar-1 atconcentration 1 nM.

In some embodiments, the present disclosure provides methods forstimulating macrophage activation and polarization toward apro-resolution state (i.e., M2) in a subject by enhancing the amount ofSPMs and/or SPM Precursor species present in a naturally occurring oilcomposition.

In one embodiment, the polyunsaturated fatty acid composition featuresmethods to elevate SPM levels in plasma in a subject by administering tothe subject an effective amount of the composition effective to elevateSPM levels in a subject.

RvD1 has been established as an activator of non-phlogistic phagocytosisin macrophages, which is an essential step for the resolution of theinflammatory response.

In some embodiments, the composition features methods to elevate RvD1levels in plasma in a subject by administering to the subject aneffective amount of the composition to elevate RvD1 level in a subject.

In one embodiment, subjects who were administered 4 capsules of acomposition containing 250 mg per capsule, as shown in FIG. 3,experienced a 53% greater elevation in plasma levels of RvD1 thansubjects who were administered a placebo.

In one preferred embodiment, the plasma elevation is produced one hourafter the administration of the capsules.

In one embodiment, the effective amount to elevate RvD1 in plasma levelsin a subject is a daily dose of at least 300 mg DHA, approximately 100mg EPA, ˜12.5 μg of 17-HDHA, and ˜10 μg of 18-HEPE.

In another preferred embodiment, the effective amount to elevate RvD1 inplasma levels in a subject is a daily dose of at least ˜1200 mg DHA,˜400 mg EPA, ˜50 μg of 17-HDHA and ˜40 μg of 18-HEPE.

In some embodiments, the polyunsaturated fatty acid composition featuresmethods for treating and resolving inflammation in a subject (e.g., ahuman being, dog, cat, horse and other animals) having a disease with aninflammatory component by administering to the subject an effectiveamount of the composition in an amount effective to activate resolutionmechanisms and thus resolve local inflammation.

In several embodiments, the diseases with an inflammatory component canbe selected from Crohn's disease, IBD, ulcerative colitis, fatty liver,wound healing, arterial inflammation, sickle-cell disease, arthritis,psoriasis, urticaria, vasculitis, asthma, ocular inflammation, pulmonaryinflammation, dermatitis, cardiovascular diseases, AIDS, Alzheimer'sdisease, atherosclerosis, cancer, type 2 diabetes, hypertension,infectious diseases, leukemia/lymphoma, metabolic syndrome, neonatology,neuromuscular disorders, obesity, perinatal disorders, rheumaticdiseases, stroke, surgical transplantation, vascular disorders,periodontal diseases, brain injury, trauma and neuronal inflammation,among others.

In some embodiments, the composition can be manufactured as apharmaceutical composition, a dietary supplement, a nutraceuticalproduct, a medical food composition, and a nutritional composition forinfant formulae and/or prenatal formulae.

In other embodiments, the composition can be used as a vaccinecoadjuvant to potentiate the immune response to an antigen and/ormodulate it towards the desired immune response.

The tumour microenvironment, orchestrated largely by inflammatory cells,is an indispensable participant in the neoplastic process, fosteringproliferation, survival and migration of the tumour cells. Now it iswell known that inflammation is a critical component of tumourprogression.

In another embodiment, due to the inflammatory component associated withcancer and the inflammation-resolving character of the composition, thecomposition can be useful as a chemotherapeutic coadjuvant.

In some embodiments, the present disclosure provides a polyunsaturatedfatty acid composition comprising about 20% to about 95%, by weight,Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount of about0.0005% to about 1%, by weight. In some embodiments, the compositionfurther comprises an acceptable carrier. In some embodiments, thecomposition is present in a capsule or other suitable dosage unit. Insome embodiments, the Omega-3 fatty acids comprise DHA and/or EPA. Insome embodiments, the DHA is present in an amount of about 30% to about45% by weight of the composition; the EPA is present in an amount ofabout 10% to about 26% by weight of the composition; the 17-HDHA ispresent in an amount of about 0.0004% to about 0.04% by weight of thecomposition; and the 18-HEPE is present in an amount of about 0.0003% toabout 0.04% by weight of the composition. In some embodiments, thecomposition further comprises DPA. In some embodiments, the compositionfurther comprises 14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a dietarysupplement, nutraceutical product, or medical food compositioncomprising a composition comprising about 20% to about 95%, by weight,Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount of about0.0005% to about 1%, by weight. In some embodiments, the compositionfurther comprises an acceptable carrier. In some embodiments, thecomposition is present in a capsule or other suitable dosage unit. Insome embodiments, the Omega-3 fatty acids comprise DHA and/or EPA. Insome embodiments, the DHA is present in an amount of about 30% to about45% by weight of the composition; the EPA is present in an amount ofabout 10% to about 26% by weight of the composition; the 17-HDHA ispresent in an amount of about 0.0004% to about 0.04% by weight of thecomposition; and the 18-HEPE is present in an amount of about 0.0003% toabout 0.04% by weight of the composition. In some embodiments, thecomposition further comprises DPA. In some embodiments, the compositionfurther comprises 14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a nutritionalcomposition for infant formulae and/or prenatal formulae comprising acomposition comprising about 20% to about 95%, by weight, Omega-3 fattyacids and 17-HDHA and 18-HEPE in a total amount of about 0.0005% toabout 1%, by weight. In some embodiments, the composition furthercomprises an acceptable carrier. In some embodiments, the composition ispresent in a capsule or other suitable dosage unit. In some embodiments,the Omega-3 fatty acids comprise DHA and/or EPA. In some embodiments,the DHA is present in an amount of about 30% to about 45% by weight ofthe composition; the EPA is present in an amount of about 10% to about26% by weight of the composition; the 17-HDHA is present in an amount ofabout 0.0004% to about 0.04% by weight of the composition; and the18-HEPE is present in an amount of about 0.0003% to about 0.04% byweight of the composition. In some embodiments, the composition furthercomprises DPA. In some embodiments, the composition further comprises14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a composition comprising about 20% to about 95%,by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amountof about 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a process forobtaining a composition as disclosed herein from an oil using a methodselected from chromatography, extraction, distillation and/or vacuumrectification. In some embodiments, the composition comprises about 20%to about 95%, by weight, Omega-3 fatty acids, and 17-HDHA and 18-HEPE ina total amount of about 0.0005% to about 1%, by weight. In someembodiments, the composition further comprises an acceptable carrier. Insome embodiments, the composition is present in a capsule or othersuitable dosage unit. In some embodiments, the Omega-3 fatty acidscomprise DHA and/or EPA. In some embodiments, the DHA is present in anamount of about 30% to about 45% by weight of the composition; the EPAis present in an amount of about 10% to about 26% by weight of thecomposition; the 17-HDHA is present in an amount of about 0.0004% toabout 0.04% by weight of the composition; and the 18-HEPE is present inan amount of about 0.0003% to about 0.04% by weight of the composition.In some embodiments, the composition further comprises DPA. In someembodiments, the composition further comprises 14-HDHA, optionally5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a process forobtaining a composition as disclosed herein from a crude oil, theprocess comprising: (i) chemically esterifying crude oil with ethanoland a basic catalyst, at a temperature of about 55° C. to about 75° C.to produce esterified oil; (ii) distilling the esterified oil, undervacuum of about 0.01 mbar to about 0.6 mbar and at a temperature ofabout 130° C. to 190° C. for about 10 seconds to about 5 minutes toseparate fatty acids shorter than 20 carbons to obtain a distilledesterified oil comprising about 20% to about 95% by weight EPA and/orDHA, and about 0.0005% to 1% by weight 17-HDHA and 18-HEPE; and (ii)subjecting the polyunsaturated fatty acid composition obtained in step(ii) to supercritical fluid extraction with CO₂ as a supercritical fluidat a temperature of about 39° C. to 46° C. and pressure of about 80 barto 115 bar to remove oxidation, decomposition and/or degradationproducts as oligomers, dimers, polymers and conjugated dienes from thecomposition. In some embodiments, step (ii) is made in several stages inseries. In some embodiments, the process further comprises distillingthe esterified oil obtained in step (ii) to remove fatty acidscontaining more than 23 carbon atoms in the corresponding fatty acidchains before step (iii). In some embodiments, the process furthercomprises bleaching the polyunsaturated fatty acid composition undervacuum with bleaching earths and diatomaceous earths. In someembodiments, the process further comprises transesterifying thepolyunsaturated fatty acid composition in ethyl ester form to obtain acomposition containing a mixture of tri-glycerides, mono-glycerides anddi-glycerides. In some embodiments, the transesterifying comprisescatalysis with one or more lipases. In some embodiments, the processfurther comprises deodorizing the composition of interest by applying acounterflow of nitrogen or water steam to the composition under vacuum.In some embodiments, the oil is obtained from fish, krill, vegetable,and/or microbes. In some embodiments, the composition comprises about20% to about 95%, by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPEin a total amount of about 0.0005% to about 1%, by weight. In someembodiments, the composition further comprises an acceptable carrier. Insome embodiments, the composition is present in a capsule or othersuitable dosage unit. In some embodiments, the Omega-3 fatty acidscomprise DHA and/or EPA. In some embodiments, the DHA is present in anamount of about 30% to about 45% by weight of the composition; the EPAis present in an amount of about 10% to about 26% by weight of thecomposition; the 17-HDHA is present in an amount of about 0.0004% toabout 0.04% by weight of the composition; and the 18-HEPE is present inan amount of about 0.0003% to about 0.04% by weight of the composition.In some embodiments, the composition further comprises DPA. In someembodiments, the composition further comprises 14-HDHA, optionally5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a process forobtaining a polyunsaturated fatty acid composition from a concentratedesterified oil comprising: (i) distilling the concentrated esterifiedoil under vacuum of about 0.01 mbar to 0.6 mbar and at a temperature ofabout 130° C. to about 190° C. for about 10 seconds to about 5 minutesto separate fatty acids having fewer than 20 carbon atoms in thecorresponding fatty acid chain to obtain a distilled esterified oilcomprising about 20% to about 95% by weight EPA and/or DHA, and about0.0005% to about 1% by weight 17-HDHA and 18-HEPE; (ii) subjecting thedistilled esterified oil obtained in step (i) to supercritical fluidextraction with CO₂ as a supercritical fluid at a temperature of about20° C. to 40° C. and at a pressure of about 80 bar to about 115 bar toremove oxidation, decomposition and degradation products as oligomers,dimers, polymers and conjugated dienes from the composition. In someembodiments, step (i) is made in several stages in series. In someembodiments, the process further comprises distilling the distilledesterified oil obtained in step (i) to remove fatty acids having morethan 23 carbon atoms in the corresponding fatty acid chain before step(ii). In some embodiments, the process further comprises bleaching thepolyunsaturated fatty acid ethyl ester composition under vacuum withbleaching earths and diatomaceous earths. In some embodiments, theprocess further comprises transesterifying the polyunsaturated fattyacid ethyl ester composition to obtain a composition containing amixture of tri-glycerides, mono-glycerides and di-glycerides. In someembodiments, the transesterifying comprises catalysis with one or morelipases. In some embodiments, the process further comprises deodorizingthe polyunsaturated fatty acid ethyl ester composition of interest byapplying a counterflow of nitrogen or water steam to the compositionunder vacuum. In some embodiments, the concentrated esterified oil isobtained from fish, krill, vegetable, and/or microbes. In someembodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a method ofincreasing phagocytic activity of macrophages in a subject, comprisingadministering to the subject a phagocytic activity enhancing amount of acomposition as disclosed herein. In some embodiments, the compositioncomprises about 20% to about 95%, by weight, Omega-3 fatty acids and17-HDHA and 18-HEPE in a total amount of about 0.0005% to about 1%, byweight. In some embodiments, the composition further comprises anacceptable carrier. In some embodiments, the composition is present in acapsule or other suitable dosage unit. In some embodiments, the Omega-3fatty acids comprise DHA and/or EPA. In some embodiments, the DHA ispresent in an amount of about 30% to about 45% by weight of thecomposition; the EPA is present in an amount of about 10% to about 26%by weight of the composition; the 17-HDHA is present in an amount ofabout 0.0004% to about 0.04% by weight of the composition; and the18-HEPE is present in an amount of about 0.0003% to about 0.04% byweight of the composition. In some embodiments, the composition furthercomprises DPA. In some embodiments, the composition further comprises14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a method ofenhancing macrophage polarization toward a pro-resolution phenotype in asubject, comprising administering to the subject a macrophagepolarization enhancing amount of a composition as disclosed herein. Insome embodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a method ofresolving inflammation associated with a disease in a subject in needthereof, comprising administering to the subject aninflammation-resolving amount of a composition as disclosed herein. Insome embodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the disease is selected fromCrohn's disease, irritable bowel disease (“IBD”), fatty liver, woundhealing, arterial inflammation, sickle-cell disease, arthritis,psoriasis, urticaria, vasculitis, asthma, ocular inflammation, pulmonaryinflammation, dermatitis, cardiovascular diseases, AIDS, Alzheimer'sdisease, atherosclerosis, cancer, type 2 diabetes, hypertension,infectious diseases, leukemia/lymphoma, metabolic syndrome, neonatology,neuromuscular disorders, obesity, perinatal disorders, rheumaticdiseases, stroke, surgical transplantation, vascular disorders,periodontal diseases, brain injury, trauma and neuronal inflammation.

In some embodiments, the present disclosure provides a method forelevating SPM levels in plasma of a human subject, comprisingadministering to the human subject a composition as disclosed herein. Insome embodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the SPM comprises RvD1.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein as a vaccine coadjuvant. In someembodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein as a chemotherapeutic coadjuvant. Insome embodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a composition asdisclosed herein for increasing phagocytic activity of macrophages in asubject. In some embodiments, the composition comprises about 20% toabout 95%, by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in atotal amount of about 0.0005% to about 1%, by weight. In someembodiments, the composition further comprises an acceptable carrier. Insome embodiments, the composition is present in a capsule or othersuitable dosage unit. In some embodiments, the Omega-3 fatty acidscomprise DHA and/or EPA. In some embodiments, the DHA is present in anamount of about 30% to about 45% by weight of the composition; the EPAis present in an amount of about 10% to about 26% by weight of thecomposition; the 17-HDHA is present in an amount of about 0.0004% toabout 0.04% by weight of the composition; and the 18-HEPE is present inan amount of about 0.0003% to about 0.04% by weight of the composition.In some embodiments, the composition further comprises DPA. In someembodiments, the composition further comprises 14-HDHA, optionally5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a composition asdisclosed herein for enhancing macrophage polarization toward apro-resolution phenotype in a subject. In some embodiments, thecomposition comprises about 20% to about 95%, by weight, Omega-3 fattyacids and 17-HDHA and 18-HEPE in a total amount of about 0.0005% toabout 1%, by weight. In some embodiments, the composition furthercomprises an acceptable carrier. In some embodiments, the composition ispresent in a capsule or other suitable dosage unit. In some embodiments,the Omega-3 fatty acids comprise DHA and/or EPA. In some embodiments,the DHA is present in an amount of about 30% to about 45% by weight ofthe composition; the EPA is present in an amount of about 10% to about26% by weight of the composition; the 17-HDHA is present in an amount ofabout 0.0004% to about 0.04% by weight of the composition; and the18-HEPE is present in an amount of about 0.0003% to about 0.04% byweight of the composition. In some embodiments, the composition furthercomprises DPA. In some embodiments, the composition further comprises14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a composition asdisclosed herein for resolving inflammation associated with a disease ina subject in need thereof. In some embodiments, the compositioncomprises about 20% to about 95%, by weight, Omega-3 fatty acids and17-HDHA and 18-HEPE in a total amount of about 0.0005% to about 1%, byweight. In some embodiments, the composition further comprises anacceptable carrier. In some embodiments, the composition is present in acapsule or other suitable dosage unit. In some embodiments, the Omega-3fatty acids comprise DHA and/or EPA. In some embodiments, the DHA ispresent in an amount of about 30% to about 45% by weight of thecomposition; the EPA is present in an amount of about 10% to about 26%by weight of the composition; the 17-HDHA is present in an amount ofabout 0.0004% to about 0.04% by weight of the composition; and the18-HEPE is present in an amount of about 0.0003% to about 0.04% byweight of the composition. In some embodiments, the composition furthercomprises DPA. In some embodiments, the composition further comprises14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the disease is selected fromCrohn's disease, irritable bowel disease (IBD), fatty liver, ulcerativecolitis, wound healing, arterial inflammation, sickle-cell disease,arthritis, psoriasis, urticaria, vasculitis, asthma, ocularinflammation, pulmonary inflammation, dermatitis, cardiovasculardiseases, AIDS, Alzheimer's disease, atherosclerosis, cancer, type 2diabetes, hypertension, infectious diseases, leukemia/lymphoma,metabolic syndrome, neonatology, neuromuscular disorders, obesity,perinatal disorders, rheumatic diseases, stroke, surgicaltransplantation, vascular disorders, periodontal diseases, brain injury,trauma and neuronal inflammation.

In some embodiments, the present disclosure provides a composition asdisclosed herein for elevating SPM levels in plasma of a human subject.In some embodiments, the composition comprises about 20% to about 95%,by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amountof about 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the SPM comprises RvD1.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein in the manufacture of a medicament forincreasing phagocytic activity of macrophages in a subject. In someembodiments, the composition comprises about 20% to about 95%, byweight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in a total amount ofabout 0.0005% to about 1%, by weight. In some embodiments, thecomposition further comprises an acceptable carrier. In someembodiments, the composition is present in a capsule or other suitabledosage unit. In some embodiments, the Omega-3 fatty acids comprise DHAand/or EPA. In some embodiments, the DHA is present in an amount ofabout 30% to about 45% by weight of the composition; the EPA is presentin an amount of about 10% to about 26% by weight of the composition; the17-HDHA is present in an amount of about 0.0004% to about 0.04% byweight of the composition; and the 18-HEPE is present in an amount ofabout 0.0003% to about 0.04% by weight of the composition. In someembodiments, the composition further comprises DPA. In some embodiments,the composition further comprises 14-HDHA, optionally 5-HETE, andoptionally 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid(“MaR1”). In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from a vegetable, a microbe, an animal, or acombination thereof. In some embodiments, the Omega-3 fatty acids,17-HDHA and 18-HEPE are obtained from fish oil, krill oil, vegetableoil, microbial oil, or a combination thereof. In some embodiments, theOmega-3 fatty acids, 17-HDHA and 18-HEPE are obtained from fish oil. Insome embodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein in the manufacture of a medicament forenhancing macrophage polarization toward a pro-resolution phenotype in asubject. In some embodiments, the composition comprises about 20% toabout 95%, by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in atotal amount of about 0.0005% to about 1%, by weight. In someembodiments, the composition further comprises an acceptable carrier. Insome embodiments, the composition is present in a capsule or othersuitable dosage unit. In some embodiments, the Omega-3 fatty acidscomprise DHA and/or EPA. In some embodiments, the DHA is present in anamount of about 30% to about 45% by weight of the composition; the EPAis present in an amount of about 10% to about 26% by weight of thecomposition; the 17-HDHA is present in an amount of about 0.0004% toabout 0.04% by weight of the composition; and the 18-HEPE is present inan amount of about 0.0003% to about 0.04% by weight of the composition.In some embodiments, the composition further comprises DPA. In someembodiments, the composition further comprises 14-HDHA, optionally5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein in the manufacture of a medicament forresolving inflammation associated with a disease in a subject in needthereof. In some embodiments, the composition comprises about 20% toabout 95%, by weight, Omega-3 fatty acids and 17-HDHA and 18-HEPE in atotal amount of about 0.0005% to about 1%, by weight. In someembodiments, the composition further comprises an acceptable carrier. Insome embodiments, the composition is present in a capsule or othersuitable dosage unit. In some embodiments, the Omega-3 fatty acidscomprise DHA and/or EPA. In some embodiments, the DHA is present in anamount of about 30% to about 45% by weight of the composition; the EPAis present in an amount of about 10% to about 26% by weight of thecomposition; the 17-HDHA is present in an amount of about 0.0004% toabout 0.04% by weight of the composition; and the 18-HEPE is present inan amount of about 0.0003% to about 0.04% by weight of the composition.In some embodiments, the composition further comprises DPA. In someembodiments, the composition further comprises 14-HDHA, optionally5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the disease is selected fromCrohn's disease, irritable bowel disease (“IBD”), fatty liver,ulcerative colitis, wound healing, arterial inflammation, sickle-celldisease, arthritis, psoriasis, urticaria, vasculitis, asthma, ocularinflammation, pulmonary inflammation, dermatitis, cardiovasculardiseases, AIDS, Alzheimer's disease, atherosclerosis, cancer, type 2diabetes, hypertension, infectious diseases, leukemia/lymphoma,metabolic syndrome, neonatology, neuromuscular disorders, obesity,perinatal disorders, rheumatic diseases, stroke, surgicaltransplantation, vascular disorders, periodontal diseases, brain injury,trauma and neuronal inflammation.

In some embodiments, the present disclosure provides a use of acomposition as disclosed herein in the manufacture of a medicament forelevating SPM levels in plasma of a human subject. In some embodiments,the composition comprises about 20% to about 95%, by weight, Omega-3fatty acids and 17-HDHA and 18-HEPE in a total amount of about 0.0005%to about 1%, by weight. In some embodiments, the composition furthercomprises an acceptable carrier. In some embodiments, the composition ispresent in a capsule or other suitable dosage unit. In some embodiments,the Omega-3 fatty acids comprise DHA and/or EPA. In some embodiments,the DHA is present in an amount of about 30% to about 45% by weight ofthe composition; the EPA is present in an amount of about 10% to about26% by weight of the composition; the 17-HDHA is present in an amount ofabout 0.0004% to about 0.04% by weight of the composition; and the18-HEPE is present in an amount of about 0.0003% to about 0.04% byweight of the composition. In some embodiments, the composition furthercomprises DPA. In some embodiments, the composition further comprises14-HDHA, optionally 5-HETE, and optionally7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (“MaR1”). Insome embodiments, the Omega-3 fatty acids, 17-HDHA and 18-HEPE areobtained from a vegetable, a microbe, an animal, or a combinationthereof. In some embodiments, the Omega-3 fatty acids, 17-HDHA and18-HEPE are obtained from fish oil, krill oil, vegetable oil, microbialoil, or a combination thereof. In some embodiments, the Omega-3 fattyacids, 17-HDHA and 18-HEPE are obtained from fish oil. In someembodiments, the Omega-3 fatty acids are in the form of free fattyacids, esters, phospholipids, mono-glycerides, di-glycerides,tri-glycerides and/or combinations thereof. In some embodiments, theOmega-3 fatty acids are in ethyl ester form. In some embodiments, the17-HDHA and 18-HEPE are in the form of free fatty acids, esters,phospholipids, mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the 17-HDHA and 18-HEPE arein the form of mono-glycerides, di-glycerides, tri-glycerides and/orcombinations thereof. In some embodiments, the SPM comprises RvD1.

EXAMPLES Example 1. Obtain an Oil Fraction Comprising DHA, EPA, 17-HDHAand 18-HEPE from Semi-Refined Marine Oil

Six compositions of polyunsaturated fatty acids containing Omega-3(20-95% by weight), 17-HDHA and 18-HEPE (about 0.0005-1% by weight) areincluded as examples of compositions of the present disclosure.

The determination of the Omega-3 content was performed using EuropeanPharmacopeia 2.4.29 method and determination of 17-HDHA and 18-HEPE wasperformed by liquid chromatography-tandem mass spectrometry employingdiagnostic transition and co-elution with synthesized deuteratedstandards of 17-HDHA and 18-HEPE.

1.1 LM03-1.

This composition was obtained from a starting material consisting onesterified concentrated marine oil.

Esterified semirefined marine oil was then injected in a continuous flowrate in a vacuum distillation unit, composed mainly for an evaporator,that works at high vacuum in this example at 0.08-0.12 mbar and hightemperature (140-144° C.), and within the time of exposure of thematerial at these conditions from 10 seconds to 5 minutes. In this stagean ethyl ester rich in fatty acids mainly shorter than C20 is distilledoff. Then, the obtained marine oil was distilled to remove the highercomponents (with more than 23 carbon atoms), working at 0.09-0.12 mbarin pressure and 140-157° C. in temperature.

These steps have been covered in two times in series.

To improve the quality of the composition and remove oxidation,decomposition and degradation products as oligomers, dimers, polymersand conjugated dienes, the composition was subjected to a supercriticalfluid extraction (SFE) by counter current extraction on a column withcarbon dioxide at supercritical conditions, working at 44.5-45.5° C. intemperature and at a pressure of 80-86 bar.

The composition obtained contained a total amount of Omega-3 of about629.9 mg/g (as FFA), DHA in an amount of about 342.1 mg/g (as FFA), EPAin an amount of about 187.6 mg/g (as FFA), 17-HDHA in an amount of about60.8 mg/kg and 18-HEPE in an amount of about 87.5 mg/kg with 1.4 mg/g ofa mixed natural tocopherols to improve the oxidative stability of theproduct. The analytical data and content of this fraction is shown inTable 1.

TABLE 1 Analysis of LM03-1 Determination Result Method SPM content17HDHA (mg/kg) 60.8 LC/MS 18HEPE (mg/kg) 87.5 LC/MS 17HDHA + 18HEPE(mg/kg) 148.3 LC/MS Fatty acid profile EPA (mg/g as FFA) 187.6 Eur. Ph.2.4.29 DHA (mg/g as FFA) 342.1 Eur. Ph. 2.4.29 Total Omega-3 (mg/g asFFA) 629.9 Eur. Ph. 2.4.29 Sum of 18:3 ω-3, 18:4 ω-3, 20:4 ω-3, 20:5ω-3, 21:5 ω-3, 22:5 ω-3, 22:6 ω-3

The analysis of the chemical status of 17-HDHA and 18-HEPE in fractionLM03.1 is shown in Table 2, where the percentages of each chemical formare expressed by weight.

TABLE 2 Distribution of chemical forms of SPMs in LM03-1 % 17-HDHA(EE)in LM03-1 88.67 % 17-HDHA in other chemical form 11.33(mono-glyceride, Di-glyceride, tri- glyceride and FFA) in LM03-1 %18-HEPE (EE)in LM03-1 87.52 % 18-HEPE in other chemical form 12.48(mono-glyceride, Di-glyceride, tri- glyceride and FFA) in LM03-1

1.2 LM03-2.

This composition was obtained from a starting material consisting onesterified concentrated marine oil.

Esterified semirefined marine oil was then injected in a continuous flowrate in a vacuum distillation unit, composed mainly for an evaporator,that works at high vacuum in this example at 0.08-0.12 mbar and hightemperature (140-144° C.), and within the time of exposure of thematerial at these conditions from 10 seconds to 5 minutes. In this stagean ethyl ester rich in fatty acids mainly shorter than C20 was distilledoff. Then, the obtained marine oil was redistilled to reduce once moretime the shorter fatty acids working at 0.09-0.12 mbar in pressure and140-157° C. in temperature. This final fraction and the obtained asdistillate in the first stage were mixed and then distilled, in twostages in series: in the first stage working at 0.08-0.12 mbar ofpressure and 140-144° C. of temperature and, in the second stage,working at 0.09-0.12 mbar in pressure and 140-157° C. in temperature.

To improve the quality of the composition and remove oxidation,decomposition and degradation products as oligomers, dimers, polymersand conjugated dienes, the composition was subjected to a supercriticalfluid extraction (SFE) by counter current extraction on a column withcarbon dioxide at supercritical conditions, working at 44.5-45.5° C. intemperature and at a pressure of 80-86 bar.

LM03-2 contained a total amount of Omega-3 of about 600 mg/g (as FFA),DHA in an amount of about 300.0 mg/g (as FFA), EPA in an amount of about100.0 mg/g (as FFA), 17-HDHA in an amount of about 50.0 mg/kg and18-HEPE in an amount of about 40.0 mg/kg with a maximum of 1.4 mg/g of amixed natural tocopherols to improve the oxidative stability of theproduct. The analytical data and content of this fraction is shown inTable 3.

TABLE 3 Analysis of LM03-2 Determination Result Method SPM content17HDHA (mg/kg) 50.0 LC/MS 18HEPE (mg/kg) 40.0 LC/MS 17HDHA + 18HEPE(mg/kg) 90.0 LC/MS Fatty acid profile EPA (mg/g as FFA) 100.0 Eur. Ph.2.4.29 DHA (mg/g as FFA) 300.0 Eur. Ph. 2.4.29 Total Omega-3 (mg/g asFFA) 600.0 Eur. Ph. 2.4.29 Sum of 18:3 ω-3, 18:4 ω-3, 20:4 ω-3, 20:5ω-3, 21:5 ω-3, 22:5 ω-3, 22:6 ω-3

1.3. LM03-3.

This composition was obtained using the process described above from astarting material consisting on esterified concentrated marine oil.

Esterified semirefined marine oil was then injected in a continuous flowrate in a vacuum distillation unit, composed mainly for an evaporator,that works at high vacuum in this example at 0.08-0.12 mbar and hightemperature (150-154° C.), and within the time of exposure of thematerial at these conditions from 10 seconds to 5 minutes. In this stagean ethyl ester rich in fatty acids mainly shorter than twenty carbonswas distilled off. Then, the obtained marine oil was redistilled toreduce once more time the shorter fatty acids working at 0.08-0.12 mbarin pressure and 155-165° C. in temperature.

To improve the quality of the composition and remove oxidation,decomposition and degradation products as oligomers, dimers, polymersand conjugated dienes, the composition was subjected to a supercriticalfluid extraction (SFE) by counter current extraction on a column withcarbon dioxide at supercritical conditions, working at 44.5-45.5° C. intemperature and at a pressure of 80-86 bar.

The composition LM03-3 obtained contained a total amount of Omega-3 ofabout 663.0 mg/g (as FFA), including DHA in an amount of about 441.7mg/g (as FFA), and EPA in an amount of about 112.7 mg/g (as FFA). LM03-3also contained significant amounts of 17-HDHA, 18-HEPE, 14-HDHA, 5-HETEand maresin 1 (MaR1), and no significant amounts of pro-inflammatory inan amount of about 65.2 mg/kg with a maximum of 1.1 mg/g of a mixednatural tocopherols to improve the oxidative stability of the product.The analytical data and content of this fraction is shown in Table 4.

TABLE 4 Analysis of LM03-3 Component Abundance wt. % SPMs/SPM 18-HEPE278550 μg/kg 0.02786 wt. % Precursors 17-HDHA 146748 μg/kg 0.01467 wt. %14-HDHA 97715 μg/kg 0.00977 wt. % maresin 1 7545 μg/kg 0.00075 wt. %15-HETE 6970 μg/kg 0.00070 wt. % 5-HETE 5474 μg/kg 0.00055 wt. % RvE32617 μg/kg 0.00026 wt. % RvD5 1241 μg/kg 0.00012 wt. % MaR1 n-3DPA 827μg/kg 0.00008 wt. % RvD6 440 μg/kg 0.00004 wt. % PD1 397 μg/kg 0.00004wt. % RvD5m-3DPA 332 μg/kg 0.00003 wt. % RvD2n-3DPA 186 μg/kg 0.00002wt. % RvE2 179 μg/kg 0.00002 wt. % RvD1 173 μg/kg 0.00002 wt. % RvE1 112μg/kg 0.00001 wt. % lipoxin B4 126 μg/kg 0.00001 wt. % RvD4 97 μg/kg0.00001 wt. % lipoxin A4 94 μg/kg 0.00001 wt. % RvD2 83 μg/kg 0.00001wt. % RvD1n-3DPA 7 μg/kg 0.00000 wt. % RvD3 5 μg/kg 0.00000 wt. % TOTALSPM/SPM 549917 μg/kg 0.05499 wt. % PRECURSORS: Pro- 12-HETE 2878 μg/kg0.00029 wt. % Inflamma- LTB4 669 μg/kg 0.00007 wt. % toriesProstaglandin E2 564 μg/kg 0.00006 wt. % Prostaglandin D2 300 μg/kg0.00003 wt. % Thromboxane B2 56 μg/kg 0.00001 wt. % Prostaglandin F_(2α)34 μg/kg 0.00000 wt. % TOTAL PRO- 4501 μg/kg 0.00045 wt. %INFLAMMATORIES: ω-3 DHA (as free fatty acid) 441.7 mg/g 44.17 wt. %Fatty Acids EPA (as free fatty acid) 112.7 mg/g 11.27 wt. % OtherOmega-3 Fatty 108.6 mg/g 10.86 wt. % Acids* TOTAL OMEGA-3 663.0 mg/g66.30 wt. % FATTY ACIDS *18:3ω-3, 18:4 ω-3, 20:4 ω-3, 21:5 ω-3, and 22:5ω-3.

1.4. LM03-4.

This composition was obtained from a starting material consisting onsemirefined marine oil.

Esterified semirefined marine oil was then injected in a continuous flowrate in a vacuum distillation unit, composed mainly for an evaporator,working at high vacuum (0.18 to 0.20 mbar) and high temperature (140° C.to 150° C.), and within the time of exposure of the material at theseconditions from 10 seconds to 5 minutes. In this stage an ethyl esterrich in fatty acids mainly shorter than twenty carbons was distilledoff. Then, the obtained marine oil was distilled to remove the highercomponents (with more than 23 carbon atoms), working at 155-172° C. and0.18 to 0.20 mbar.

The obtained fraction was distilled in a second stage, working in thefirst stage 0.08-0.12 mbar of pressure and 140-144° C. of temperatureand, in the second stage, working at 0.09-0.12 mbar in pressure and140-157° C. in temperature.

Intermediate product was then processed in a urea complexation step toreduce the content of saturated and monounsaturated fatty acids. Ureawas solved in ethanol and intermediate product is added. When, urea wasprecipitated from this solution, by forming adduct products with mainlysaturated and monounsaturated ethyl esters. The urea adduct was removedby filtration, and ethanol was recovered by distillation. The resultingethyl esters were washed with demineralized water. This step can be donein two stages in series.

A bleaching step was performed then, to control and reduce urea traces.

The composition obtained contained a total amount of Omega-3 of about870.0 mg/g (as FFA), DHA in an amount of about 345.0 mg/g (as FFA), EPAin an amount of about 425.0 mg/g (as FFA), 17-HDHA in an amount of about56.2 mg/kg and 18 HEPE in an amount of about 96.4 mg/kg with a maximumof 4.8 mg/g of a-tocopherol to improve the oxidative stability of theproduct. The analytical data and content of this fraction is shown inTable 5.

TABLE 5 Analysis of LM03-4 Determination Result Method SPM content17HDHA (mg/kg) 56.2 LC/MS 18HEPE (mg/kg) 96.4 LC/MS 17HDHA + 18HEPE(mg/kg) 152.6 LC/MS Fatty acid profile EPA (mg/g as FFA) 425.0 Eur. Ph.2.4.29 DHA (mg/g as FFA) 345.0 Eur. Ph. 2.4.29 Total Omega-3 (mg/g asFFA) 870.0 Eur. Ph. 2.4.29 Sum of 18:3 ω-3, 18:4 ω-3, 20:4 ω-3, 20:5ω-3, 21:5 ω-3, 22:5 ω-3, 22:6 ω-3

1.5. LM03-5

This composition was obtained from a starting material consisting onsemirefined marine oil.

Semirefined marine oil was esterified to form ethyl esters, bycontacting with ethanol and a basic catalyst (EtONa), at 55-65° C. aknown quantity of ethanol and catalyst are added to the reactor. Afterthe reaction time (2-4 hours) concluded, the excess of additives wereevaporated. Then, a final washing step was included in order toneutralize the semirefined marine oil esterified.

The composition obtained LM03-5 contained a total amount of Omega-3 ofabout 316.0 mg/g (as FFA), DHA in an amount of about 104.0 mg/g (asFFA), EPA in an amount of about 152.0 mg/g (as FFA), n-3 DPA in anamount of about 19.0 mg/g (as FFA) 17-HDHA in an amount of about 14.6mg/kg and 18-HEPE in an amount of about 35.8 mg/kg. The analytical dataand content of this fraction is shown in Table 6.

TABLE 6 Analysis of LM03-5 Determination Result Method SPM content17HDHA (mg/kg) 14.6 LC/MS 18HEPE (mg/kg) 35.8 LC/MS 17HDHA + 18HEPE(mg/kg) 50.4 LC/MS Fatty acid profile EPA (mg/g as FFA) 152.0 Eur. Ph.2.4.29 DHA (mg/g as FFA) 104.0 Eur. Ph. 2.4.29 DPA (mg/g as FFA) 19.0Eur. Ph. 2.4.29 Total Omega-3 (mg/g as FFA) 316.0 Eur. Ph. 2.4.29 Sum of18:3 ω-3, 18:4 ω-3, 20:4 ω-3, 20:5 ω-3, 21:5 ω-3, 22:5 ω-3, 22:6 ω-3

1.6. LM03-6

This composition was obtained from a starting material consisting onesterified concentrated marine oil.

Esterified semirefined marine oil was then injected in a continuous flowrate in a vacuum distillation unit, composed mainly for an evaporator,that works at high vacuum in this example at 0.08-0.12 mbar and hightemperature (140-144° C.), and within the time of exposure of thematerial at these conditions from 10 seconds to 5 minutes. In this stagean ethyl ester rich in fatty acids mainly shorter than C20 was distilledoff. Then, the obtained marine oil was distilled to remove the highercomponents (with more than 23 carbon atoms), working at 0.09-0.12 mbarin pressure and 140-157° C. in temperature. These steps have beencovered in two times in series

To improve the quality of the composition and remove oxidation,decomposition and degradation products as oligomers, dimers, polymersand conjugated dienes, the composition was subjected to a supercriticalfluid extraction (SFE) by counter current extraction on a column withcarbon dioxide at supercritical conditions, working at 44.5-45.5° C. intemperature and at a pressure of 80-86 bar.

The composition obtained was subjected to a step of transesterificationto obtain a composition containing a mixture of tri-glycerides andpartial glycerides (mono-glycerides and di-glycerides).

The reaction of transesterification was catalyzed by lipases at atemperature of about 50° C. to about 70° C. under vacuum.

The obtained fraction was treated under vacuum with high temperaturesand a countercurrent flow of steam (nitrogen) to remove the volatilecomponents and improve the odour, if they are present. These volatilecomponents were solved into the steam and flow out the deodorizationequipment.

The composition obtained LM03-6 contained a total amount of Omega-3 ofabout 635.0 mg/g (as FFA), DHA in an amount of about 189.0 mg/g (asFFA), EPA in an amount of about 342.0 mg/g (as FFA), 17-HDHA in anamount of about 126.8 mg/kg and 18-HEPE in an amount of about 95.2 mg/kgwith a maximum of 4 mg/g of a mixed natural tocopherols to improve theoxidative stability of the product. The analytical data and content ofthis fraction is shown in Table 7.

TABLE 7 Analysis of LM03-6 Determination Result Method SPM content17HDHA (mg/kg) 126.8 LC/MS 18HEPE (mg/kg) 95.2 LC/MS 17HDHA + 18HEPE(mg/kg) 222.0 LC/MS Fatty acid profile EPA (mg/g as FFA) 342.0 Eur. Ph.2.4.29 DHA (mg/g as FFA) 189.0 Eur. Ph. 2.4.29 Total Omega-3 (mg/g asFFA) 635.0 Eur. Ph. 2.4.29 Sum of 18:3 ω-3, 18:4 ω-3, 20:4 ω-3, 20:5ω-3, 21:5 ω-3, 22:5 ω-3, 22:6 ω-3

Example 2. Study of Regulation of Phagocytosis In Vitro Employing theSpectrophotometric Evaluation of Phagocytized Zymosan Bioparticles inPMA-Stimulated THP-1 Cells

Macrophages play a central role in inflammation and host defense againstmicroorganisms, and also participate actively in the resolution ofinflammation after alternative activation from pro-inflammatorymacrophages switched towards anti-inflammation (M2-like phenotype).

To determine the capacity of promoting a macrophage pro-resolutionphenotype (i.e. M2) and therefore to foster the resolution ofinflammation, several oils were tested in a human macrophage modeladapted by López Vicario C from a method previously described (Titos etal., 2011) and compared with Mar-1 (the term Maresins is coined fromMacrophage mediator in resolving inflammation), an established activecompound in resolution of the inflammation.

THP-1 cells were cultured in a 96-well black plate at a density of3.5×104 cells/well in 200 μl RPMI 1640 medium (10% FBS). Cells weredifferentiated into macrophages with 50 ng/ml of phorbol 12-myristate13-acetate (PMA) for 2 days and then, maintained with fresh RPMI medium(10% FBS) for 1 day. Then the macrophages were washed by aspiration ×2with sterile DPBS−/− and pretreated for 15 minutes with 150 μl of thecompounds to be tested or vehicle (ethanol) using RPMI without phenolred (1% FBS). Each sample was tested in duplicate or triplicate.

The compounds tested in this macrophage model were:

-   -   Maresin 1 (from Cayman Chemicals)    -   LM03-1 (a total amount of Omega-3 of about 629.9 mg/g (as FFA),        DHA in an amount of about 342.1 mg/g (as FFA), EPA in an amount        of about 187.6 mg/g (as FFA), 17-HDHA in an amount of about 60.8        mg/kg and 18-HEPE in an amount of about 87.5 mg/kg with 1.4 mg/g        of a mixed natural tocopherols—3624 EE (about 33.0% EPA ethyl        ester and about 22% DHA in ethyl ester form expressed as FFA)    -   Omacor (about 42.5% EPA ethyl ester and about 34.5% DHA in ethyl        ester form expressed as FFA)

The concentration of Mar-1 used in the experiment was 1 nM, which hasbeen determined the concentration with maximum activity in phagocyticactivation for this compound in previous experiments using this model(data not shown). The oils tested, LM03-1, 3624 EE and Omacor, werediluted 1/10³ for use in the experiment.

After 15 minutes of pretreatment with the compounds to be tested, 50 μlof opsonized zymosan bioparticles (Molecular Probes; Z-2841; ratiocell/bioparticles=1/10) were added to each well of the plate (Finalvolume=200 μl) and mixed.

The macrophages were incubated at 37° C. for 60 minutes and then theplate was centrifuged for 5 minutes at 400 g at room temperature andsupernatant was discarded by aspiration.

The macrophages were washed with sterile DPBS−/− before adding 100 μl ofTrypan Blue Solution (diluted at 1:10 in sterile DPBS−/−) to quenchfluorescence of bioparticles bound to the outside of the cell.

Then, the macrophages were incubated for 2 minutes at room temperatureand centrifuged for 5 minutes at 400 g at room temperature and theexcess of Trypan Blue was aspirated.

The fluorescent intensity of each sample was read in the fluorescencemicroplate reader FLUOstar OPTIMA (in a Costar 96 microplate) andaverage fluorescence intensity values were calculated to obtainphagocytosis response to the effector.

Maresin-1, a very potent inducer of macrophage phagocytosis was used asa reference. 100% activity was assigned to compare the effect overphagocytic activity of the other compounds tested.

The LM03-1 oil, as shown in FIG. 2, activated macrophage activationreaching more than 55% of the activity of Mar-1 (pure compound) inmacrophage activation. LM03-1 oil fraction enhanced macrophage activityand polarization toward a pro-resolution phenotype (I.E. M2) due to itsSPMs. The results obtained in this model indicate the resolutioncapacity of LM03-1 oil fraction.

The other products tested in this model, 3624 EE and Omacor, did notactivate phagocytosis activity much more, instead, these compoundsdiminished the phagocytic activity of macrophages in this model.

Example 3. Study of Plasma Levels of RvD1 During a 1 Hour Period afterIngesting a Composition Containing DHA, EPA, 17-HDHA, and 18-HEPE

To determine the variation in plasma levels of RvD1 after ingest a ethylester Omega-3 oil fraction containing EPA, DHA, 17-HDHA and 18-HEPE(LM03-2 oil fraction obtained in Example 1) compared with a placebo, aclinical study was designed over N=10 subjects administered LM03-2composition and compared with N=5 subjects ingesting a placebo.

The composition of LM03-2 used in this clinical trial contained a totalamount of Omega-3 of about 600 mg/g (as FFA), DHA in an amount of about300.0 mg/g (as FFA), EPA in an amount of about 100.0 mg/g (as FFA),17-HDHA in an amount of about 50.0 mg/kg and 18-HEPE in an amount ofabout 40.0 mg/kg.

The subject characteristics of the study appear in Table 8.

TABLE 8 Subject characteristics of the study Variable LM03-2 (n = 10)Placebo (N = 5) P-value Age (y) 46.0 ± 2.4 51.4 ± 4.5 0.259 Height (m) 1.66 ± 0.01  1.69 ± 0.03 0.379 Weight (kg) 90.0 ± 5.6 88.5 ± 6.3 0.870BMI 32.6 ± 1.9 31.2 ± 2.2 0.657 CRP (mg/l) 5.25 ± 1.2 5.84 ± 1.3 0.772

Subjects ingested 4 capsules (250 mg each capsule) of LM03-2 or placeboat 8:00 am, blood samples were collected before ingestion of thecapsules and 15 min, 30 min and one hour post-ingestion.

LM03-2 composition of each capsule is approximately 75 mg DHA, 25 mgEPA, and SPM precursors 17-HDHA (17.5 μg) and 18-HEPE (10 μg).

RvD1 has been identified as an activator of non-phlogistic phagocytosisin macrophages, which is an essential step for the resolution of theinflammatory response. The blood samples were analyzed for plasma RvD1levels and results are shown in FIG. 3. The increase of RvD1 levels insubjects one hour after the ingestion of 4 capsules of LM03-2composition is 53% higher than the level just after the ingestion of thecomposition in these subjects, instead the levels of RvD1 one hour laterin subjects that ingested a placebo raised a 5% over their initialvalue.

This results show the capacity of the composition LM03-2 to elevate RvD1levels in plasma.

Example 4. Mice Subcutaneous Anti-Inflammatory Activity Trial

To determine the anti-inflammatory activity of different oils, threeoils were tested for their anti-inflammatory activity and compared withthe activity of indomethacin (a nonsteroidal anti-inflammatory drug) ina subcutaneous in vivo model of inflammation using CD1 white male mice,adult (30-35 grams weight).

N=5 mice were used in each group tested with an oil fraction group andN=4 mice were used in control and indomethacin groups.

A prototypical inflammatory agent, LPS (lipopolysaccharide from theouter coat of bacteria) was injected subcutaneously as a single dose (5mg per kg in a 200 μL volume) in the dorsal hind flank to createinflammation in mice.

Thirty minutes prior to the administration of LPS, 100 μL of vehiclecontrol (PBS phosphate buffer saline), indomethacin, or one of the oilfractions were administered by gavage.

Neutrophil infiltration into the site of inflammation was measurednon-invasively by bioluminescence emitted by conversion of luminol bythe neutrophil enzyme myeloperoxidase. 150 μL of a suspension of luminolin PBS (50 mg/I) was administered to the mice and the image was takenafter 15 minutes of the luminol injection.

The bioluminescence was measured using an IVIS-lumina equipment(Perkin-Elmer, Tres Cantos, Madrid, Spain).

The method used generates reproducible bioluminescence measurements ofneutrophil activity in order to be able to measure statisticallysignificant changes in neutrophil activity upon administration ofdifferent oil fractions and indomethacin.

The oils fractions tested in the experiments are as follows:

-   -   Krill: Tri-glyceride and phospholipids form (about 14.4% EPA and        7.4% DHA in weight expressed as FFA).    -   LM03-3 containing a total amount of Omega-3 of about 663.0 mg/g        (as FFA), DHA in an amount of about 441.7 mg/g (as FFA), EPA in        an amount of about 112.7 mg/g (as FFA), and about 550 mg/kg of        total SPMs/SPM Precursors, including 17-HDHA in an amount of        about 146 mg/kg and 18-HEPE in an amount of about 279 mg/kg.    -   Algae: Tri-glyceride form (about 0.2% EPA and 39.7% DHA in        weight expressed as FFA).

The results for the analysis of bioluminescence for each oil fractioncompared with control and indomethacin are shown in two figures percompound tested, measure the total flux of the bioluminescence radiation(net radiance photons/sec) and the evolution of medium radiance(photons/sec/cm²/sr) along time.

For krill oil, the response shown in FIGS. 4A and 4B was similar to thecontrol and even over the control at 180 minutes. The response comparedwith the indomethacin was 37% higher, showing a significant value ofp<0.02. As shown in FIGS. 4A and 4B, krill oil was not significantlyanti-inflammatory in this model.

The results detailed in FIGS. 5A and 5B, probe the anti-inflammatorycharacter of fraction LM03-3. LM03-3 oil shows a clear anti-inflammatoryeffect at 90, 180 and 360 minutes with a quick response at 90 min andthe effect is maintained during the 6 hours that last the experiment.This is the only tested oil that shows this quick and long effect.

At time 180 minutes, the LM03-3 oil had the maximum difference comparedto the control, with the total flux (photons/sec) 36% below the control(p<0.13).

As shown in FIG. 6A, the total flux at time 180 min the value(photons/sec) for the Algae oil is 37% higher than the positive controlindomethacin with a significant value of p<0.02.

However this difference between algae oil and the indomethacin decreasebeing at 360 min close to the indomethacin.

Example 5. Encapsulation of a Composition Containing EPA, DHA, 17-HDHAand 18-HEPE in a Capsule

The oil fraction LM03-2 obtained in example 1 was encapsulated in anoval enteric soft gelatin shell.

The specifications of encapsulated LM03-2 composition in a capsule areshown in Table 9.

TABLE 9 Specifications of LM03-2 encapsulated Capsule weight 423 mgContain weight 256 mg Weight variation 97-102% Specific gravity 0.75g/mL Gastric disintegration Conform Intestinal disintegration <15 minTotal acidity 1.51 mg KOH/g Cholesterol 0.33 mg/capsule Total Fat 256mg/capsule Total Omega-3 169 mg/capsule EPA 75 mg/capsule DHA 25mg/capsule 17-HDHA 12.5 mg/capsule 18-HEPE 10 mg/capsule

Example 6. Emulsification of a Composition Containing EPA, DHA, 17-HDHAand 18-HEPE as Delivery Form

The LM03-6 oil fraction obtained in Example 1 was mixed withdemineralised water, emulsifiers, preservatives, stabilizers, naturalflavours, pH controllers and sweeteners to provide an oral lipidicemulsion formed by a ordered network. This reaction is made at roomtemperature in absence of oxygen, by a previous blanketing step. At theend of the process, a pasteurization step is performed asmicrobiological control.

The specification of the emulsified LM03-6 composition is shown in Table10. The typical composition of the emulsified LM03-6 packed in a sachetis shown in Table 11.

TABLE 10 Specifications of emulsified LM03-6. Total Omega-3 (as FFA)Min. 50 mg/g Min. 5 wt. % EPA (as FFA) Min. 10 mg/g Min. 1 wt. % DHA (asFFA) Min. 30 mg/g Min. 3 wt. % 17-HDHA Min. 5 mg/kg Min. 0.0005 wt. %18-HEPE Min. 3 mg/kg Min. 0.0003 wt. % 17-HDHA + 18-HEPE Min. 8 mg/kgMin. 0.0008 wt. %

TABLE 11 Specifications of emulsified LM03-6 packed in a sachet. Sachet15 ml weight 14.97 g Total Omega-3 (as FFA) Min. 900 mg/sachet Min.6.012 wt. % EPA (as FFA) Min. 150 mg/sachet Min. 1.002 wt. % DHA (asFFA) Min. 450 mg/sachet Min. 3.006 wt. % 17-HDHA Min. 75 μg/sachet Min.0.0005 wt. % 18-HEPE Min. 60 μg/sachet Min. 0.0004 wt. % 17-HDHA +18-HEPE Min. 135 μg/sachet Min. 0.0009 wt. %

1-18. (canceled)
 19. A composition comprising: docosahexaenoic acid(“DHA”) an amount of about 20% to about 95%, by weight, of thecomposition; and 17-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid(“17-HDHA”), 14-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid(“14-HDHA”), 15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid(“15-HEPE”), and 18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid(“18-HEPE”), wherein the 18-HEPE is present in an amount of 0.02% toabout 1%, by weight, of the composition and the 17-HDHA is present in anamount of 0.01% to about 1%, by weight, of the composition.
 20. Thecomposition of claim 19, wherein the composition is present in a capsuleor in an emulsion.
 21. The composition of claim 19, wherein thecomposition further comprises eicosapentaenoic acid (“EPA”).
 22. Thecomposition of claim 19, wherein the DHA, 14-HDHA, 15-HEPE, 17-HDHA,and/or 18-HEPE are present in the composition as a free fatty acid,ester, phospholipid, mono-glyceride, di-glyceride, tri-glyceride, orcombinations thereof.
 23. A dietary supplement, nutraceutical product,nutritional composition for infant formula and/or prenatal formula,pharmaceutical composition, vaccine coadjuvant, chemotherapeuticcoadjuvant or medical food composition comprising the composition ofclaim
 19. 24. The composition of claim 19, wherein the 18-HEPE ispresent in an amount of about 0.03%, by weight, of the composition. 25.The composition of claim 19, wherein 14-HDHA is present in an amount ofabout 0.005% to about 0.1%, by weight, of the composition.
 26. Thecomposition of claim 25, wherein the 14-HDHA is present in an amount ofabout 0.01%, by weight, of the composition.
 27. A method of treatinginflammation comprising administering the composition of claim 19 to asubject having inflammation.
 28. A composition comprising: DHA an amountof about 20% to about 95%, by weight, of the composition; and 17-HDHA,18-HEPE, 14-HDHA, and 12-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid(“12-HEPE”), wherein the 18-HEPE is present in an amount of 0.02% toabout 1%, by weight, of the composition and the 17-HDHA is present in anamount of 0.01% to about 1%, by weight, of the composition.
 29. Thecomposition of claim 28, wherein the composition is present in a capsuleor in an emulsion.
 30. The composition of claim 28, wherein thecomposition further comprises EPA.
 31. The composition of claim 28,wherein the DHA, 14-HDHA, 12-HEPE, 17-HDHA, and/or 18-HEPE are presentin the composition as a free fatty acid, ester, phospholipid,mono-glyceride, di-glyceride, tri-glyceride, or combinations thereof.32. A dietary supplement, nutraceutical product, nutritional compositionfor infant formula and/or prenatal formula, pharmaceutical composition,vaccine coadjuvant, chemotherapeutic coadjuvant or medical foodcomposition comprising the composition of claim
 28. 33. The compositionof claim 28, wherein the 18-HEPE is present in an amount of about 0.03%,by weight, of the composition.
 34. The composition of claim 28, wherein14-HDHA is present in an amount of about 0.005% to about 0.1%, byweight, of the composition.
 35. The composition of claim 34, wherein the14-HDHA is present in an amount of about 0.01%, by weight, of thecomposition.
 36. A method of treating inflammation comprisingadministering the composition of claim 28 to a subject havinginflammation.